Evaluation of transected spinal cord regeneration in the rat

EXPERIMENTAL

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Evaluation of Transected Spinal Cord Regeneration in the Rat J. C. DE

LA

TORRE, P. K. HILL,M. AND J. C. PARKER,

GONZALEZ-CARVAJAL, JR.’

Division of Neurosurgery. Ottawa General Hospital and University of Ottawa Health Sciences, Ottawa, Ontario KIH gM5, Canada; Neurology Section, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709; the University of Miami, Miami, Florida 33141; and the University of Tennessee School of Medicine, Knoxville, Tennessee 37920 Received August 31, 1983; revision received November 14, 1983 Rat spinal cords were subjected to a 200 g/cm force acceleration injury at TIO. Ten days later, the cords were totally transected at TIO and the rats separated into two groups: group C (controls) had the spinal cord realigned end-to-end, group X had 3 mm trimmed from proximal and distal cord stumps and a semifluid collagen’ matrix (CM) bioimplant was inserted in the gap. The CM polymerized to a firm gel at body temperature within 2 h. All rats were maintained 90 days posttransection (dpt). At 90 dpt, they wem examined for local spinal cord blood tlows, somatosensory evoked potentials, and a neurological evaluation. After killing, the cords were processed for electron and light microscopy and monoamine histofluorescence. The results indicated that CM can support the growth of central neurites, fibroblasts, and an adequate anastomotic network of blood vessels.Control scar tissue does not promote the presence of nerve fibers and blood vesselsto the extent observed in the CM. Somatosensory evoked potential early waveforms were present in CM-bioimplanted rats but not in controls. No rat regained walking ability at 90 dpt but muscle tone and strength appeared better in CM-implanted than in control rats. We conclude that a CM bridge can provide a well vascularized, relatively nonhostile environment for central neurites and catecholaminergic axons extending from the proximal spinal cord tissue across the CM bridge and into the distal stump.

Abbreviations: CCV-catecholamine-containing varicosity, CM-collagen matrix, dpt-days posttransection, SEP-somatosensory evoked potentials, lSCBF-local spinal cord blood flow. ’ This work was made possible by a gift from the Ronald Purer Foundation, Inglewood, California and a grant from the American Paralysis Association, McLean, Virginia. The authors are grateful to Ms. Ruth Daly for the illustrations. Please send reprint requests to Dr. de la Torre, Division of Neurosurgery, Ottawa General Hospital, 451 Smyth Rd., Ottawa, Ont. KlH 8M5, Canada. 188 0014-4886184 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.

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INTRODUCTION Windle and Chambers (32) first described in a systematic fashion the morphologic damage associated with sensory-motor loss that follows complete transection of a mammalian spinal cord. Fifty years earlier, Rambn y Cajal (26) reported that transected axons could grow short distances but, as a rule, they did not extend across the lesion site nor reconnect synaptically with distal target tissue. Recent studies attempted to correct this problem by implanting tissue autografts to bridge the transected spinal cord stumps in order to provide a scaffold for regenerating proximal neurites. Those autografts have been taken from peripheral nerve (5, 29, 30) muscle (1, 2), fetal cells (23, 24), and omentum (16). In this report, a nontissue, cell-free bovine collagen matrix (CM, Collagen Corp., Palo Alto, Calif.) was used to bridge the gap between cut proximal and distal cord stumps. Preliminary observation found that the CM could provide a favorable environment to growing blood vessels and catecholaminergic axons when implanted in rat brain after cortical ablation (8). The results obtained demonstrated that use of the CM in transected spinal cord may similarly provide a bridge for anastomotic blood vessels, migrating fibroblasts, and growth of central neurites from the host tissue. MATERIALS

AND

METHODS

Thirty-two male, Long-Evans hooded rats weighing 375 to 475 g were anesthetized with sodium pentobarbital (21 mg/kg, i.p.) followed 10 min later by ketamine (60 mg/kg, i.m.). A laminectomy was done between T8 and T 12 using a l-mm burr connected to an air drill. The procedure was aided using a surgical microscope. To facilitate the laminectomy, muscle attachments to the transverse processes were cut bilaterally from T9 to T 11, and retracted laterally by sutures. The laminae were then cut by gently drilling at the level of the neural arches bilaterally. The supraspinous muscles were cut with fine scissors at T9 to Tl 1 and the dorsal laminae with their spinous processes were removed as a single canopy exposing about 2 cm of spinal cord. This approach exposed the dorsal spinal cord virtually without damage to the cord tissue or its vasculature. While still under anesthesia, 26 rats were subjected to a 200 g/c force injury at TlO using a modification of the Freeman and Wright (15) drop weight method (10). This injury causes permanent paralysis in rats (9, 11). The trauma is accompanied by immediate subdural hemorrhage and considerable tissue damage. Ten days after the injury, the rats were anesthetized as before and underwent bilateral rhizotomy between T 10 and T 11. To minimize tissue swelling, hemorrhage, and prevent blade compression of the tissue, all spinal cords were cooled 15 min by ice-cold sterile saline irrigation.

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The cords were slightly raised by passing a curved, 30-gauge, blunted needle underneath extradurally, and transected with a thin Gillette blade in a gentle see-saw motion. Ice-cold saline irrigation was continued for 30 min. After hemostasis, the two cord stumps were gently picked up with tissue forceps to verify complete transection. Using the surgical microscope, the severed cord was examined again to assure that no anterior or lateral fibers remained uncut. Rats were then randomly separated into two groups. Group C (control) had the spinal cord approximated in close end-to-end juxtaposition. Group X (collagen matrix) had a total of 3 mm of spinal cord tissue trimmed from the proximal and distal stumps using a new Gillette blade while the cord was irrigated with ice-cold saline. The gap was filled with sterile, semifluid collagen matrix (CM). The CM has the physical property of remaining semifluid at 0 to 4°C. It polymerizes within 2 h to a firm, opalescent gel at 37°C (body temperature). The gel formed a tight, physical continuity with the proximal-distal cord stumps. Postoperative care followed a protocol as described elsewhere (13) and was critical in the management of the rats during their recovery. Rats with no bladder control had urine expressed by CredC’s method (manual pressure) twice daily until voluntary micturition was regained. Any urinary, gastrointestinal, or skin problem was immediately treated with specific therapy ( 12). Six sham-operated rats (group S) had laminectomies without apparent injury to the spinal cord and served as noninjury controls. All rats were observed for 90 days after cord transection. Any rat that did not survive the 9 days was excluded from the study. The mortality rate during this period was 2.8%. On day 90, all rats had a neurological evaluation of motor behavior, muscle tone, and function of the rear limbs. Sensory function was tested mechanically using pin-prick stimuli. Sensory afferent neuronal conduction across the lesion was tested using bilateral electrical stimulation of the femoral nerves and skull electrode recordings of somatosensory evoked potentials (SEP). Local spinal cord blood flow (ISCBF) was recorded prior to killing to evaluate regional flows near, at the lesion site, and inside the CM bridge or scar tissue (Fig. 1). Somatosensory Evoked Potentials. (SEP). The SEP were recorded by isolating both sides of a posterior division of the femoral nerve below the injury site (10). Two Teflon-insulated platinum wires bared at the tips were lightly hooked around the nerve, anchored to the muscle fascia with a stay suture and square-wave electrical impulses of 0.1 ms duration at 0.1 ms delay given every 0.5 s. The pulses were generated by a Grass SD-9 stimulator connected to a constant current unit. The voltage was adjusted to elicit a small twitch in the paw. Control SEP peaks were first determined in each rat by median nerve stimulation (above the lesion) in the forepaws. Cortical signals from

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D FIG. 1. Microekctrode placement for measurement of local spinal cord blood flows (ISCBF) using the hydrogen clearance method. Mean differences in ISCBFs were seen between collagen matrix (CM, group X) and scar tissue (controls) in distal (D), proximal (P), and center of the graft or scar (C). Mean flows were 190% higher at C in grafted rats than in controls. Flows were 66 to 88% higher 2 mm from C in grafted rats. No ISCBF differences were seen 5 mm from the lesion site. All flow values are expressed as ml/ 100 g tissue/min. See text for technical details of electrode placement. Local Spinal Cord Blood Flows (x f SE)

CM Control *p=

A 5 mm distal

B 2 mm distal

51 +4 54 * 3

51 * 5* 32 + 3

C CMorscar 29 + 6* IO f 4

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recording skull electrodes were amplified lo5 times with a frequency response of 0.3 to 3 KHz (-6 dB) using Grass P-5 11J preamplifiers powered by a Grass RPS- 107. The signals were triggered in a Dagan 4800 computer averager and 128 responses were averaged using an analysis time of 125 to 250 ms. The summated responses were displayed in a Tektronix 922R oscilloscope. Electroencephalographic activity was monitored from the skull electrodes on a separate memory oscilloscope. Retransection of the cord above the lesion was done prior to killing in four rats from each group and SEPs were again tested using the same parameters described above. Local Spinal Cord Blood Flow (1SCBF). Spinal cord blood flows were measured from rat tissue that was later processed for SPG, EM, or silver stains. The rats were anesthetized as if for surgery and the cord exposed for

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four levels, with TlO as its center. Small cuts were made in the dura using a No. 11 blade aided by a dural hook. Microthin, light weight, insulated platinum wires measuring 10 pm at the tip and connected to 8-pm copper wires were mechanically inserted into the cord tissue using stereotaxic electrode carriers according to our floating clamp technique (18). The microelectrodes were inserted 2 and 5 mm proximal and distal to the center of the CM in group X or the tissue scar (group C) and to a depth of 1.2 mm from the dural surface and 1.O mm from the midline. The rats were exposed 10 s to hydrogen gas inhalation and hydrogen washout curves were recorded on a 4-channel Rikadenki penwriter (RV-12 type, Meguro-ku, Tokyo, Japan). The 1SCBF was calculated using the initial 2-min slope excluding the first 40 s according to the Fick principle (18). Morphological

Examination

Twelve rats from groups C and X were anesthetized as if for surgery and were pefused intracardially with 150 ml heparinized normal saline followed by 500 ml phosphate-buffered saline (20). Perfusion flow pressures were set at 170 mm mercury (11,20). The spinal cords were removed and processed for electron and light microscopy. Neuritic processes from each cord were examined using Bodian’s silver stains (4). Fourteen rats from groups C and X received 250 mg/kg, i.p. of the monoamine oxidase inhibitor, nialamide, 2 h before being killed, to enhance tissue catecholamine fluorescence in the spinal cord tissue. Their spinal cords were quickly removed without tissue perfusion and processed for catecholamines using the SPG histofluorescence method (7, 14). RESULTS Morphological

Examination

All six sham-operated control rats (group S) recovered from the laminectomy without significant neurological deficits. After 90 days those rats were processed for morphological and neurophysiological tests following the same procedure as for groups C and X. Group S rats did not differ morphologically or physiologically from the baselines established for intact control rats. Bodian Silver Stains. Numerous fibroblasts and fibroblastic processes were seen within the CM, with the greatest density at the proximal end and the least density toward the distal stump. The fibroblastic processes were distinguished from neurites by the former’s smoother surface and larger diameter compared with the neuritic processes. Examination of the tissue and scoring was done by one of us without knowledge of the treatment received. A scattering of microcysts measuring approximately 30 to 50 pm was seen near the proximal tissue but generally not at the junction with the CM. Numerous blood vessels, from capillaries to large arterioles and venules, were

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seen throughout the CM. Perfusion of the cord tissue resulted in the CM loosing its pink color and appearing blanched much like the cord tissue itself. The number of blood vessels and fibroblasts present within the scar fibers was considerably less compared with the CM of the treated tissue. A moderate number of microcysts measuring 50 to 80 pm were seen predominantly near the proximal-scar tissue junction and a few at the distal-scar tissue interface. A semiquantitative estimate of the number of neurites seen in the scar tissue of CM as it interfaced with the proximal and distal cord tissue was scored as follows: O-no neurites, + 1-one to three neurites per high-power field, +2-four to eight neurites per high-power field, +3-more than eight neurites per high-power field. On the basis of this scale, control group C had 0 neurites at the proximal or distal interface with the scar fibers. Group X (collagen matrix bridge) had on the average, +2 and +l neurites on the proximal and distal tissue junction, respectively, which interfaced with the CM bridge. Electron Microscopy. Ultrastructural examination of the tissue by one of us without prior knowledge of the treatment showed that near the collagen scar in group C rats, a few islands of unmyelinated and myelinated axons were found. The majority of myelinated axons were associated with a Schwann cell. Few blood vessels and fibroblasts were noted within the scar region. In addition, the collagen scar fibers appeared more oriented compared with the CM fibers (Fig. 2B). Group X rats showed a moderate number of myelinated and unmyelinated axons throughout the CM. Most myelinated axons were associated with a Schwann cell. A moderate number of blood vessels and fibroblasts were seen throughout the CM with a greater density distribution toward the proximal stump. Most capillaries contained endothelial cells and tight junctions and appeared morphologically identical to central capillaries in the brain. Rarely, a fenestrated capillary (noncentral) would be seen within the CM. The CM fibers appeared to have a random, loosely meshed orientation and appeared less densely packed than the control scar fibers (Fig. 2A, B). Catecholamine Histofluorescence. Ninety days after spinal cord transection, 14 rats (8 in group X, 6 in group C) had the unfixed cords removed at T i0 for three segments in a proximodistal direction. The CM or scar tissue was cut at the center and the tissue was labeled “proximal” or “distal.” Horizontal cryostat sections (16 pm) were prepared. The tissue was examined for histofluorescence of catecholamines using the SPG method (7, 14). Figure 3, shows the typical group C findings in the proximal and distal regions that interfaced with the scar tissue fibers. Scar tissue fibers were seen entering the cord tissue but no catecholamine-containing varicosities (CCVs) were found near its junction with the cord tissue nor within the scar tissue. Moreover, no CCVs were seen in the distal cord tissue within the two levels

FIG. 2. Ultrastructure of collagen scar fibers in a control (A) and a rat with a CM bioimplant (B). Note orientation of scar fibers into scrpiginous bundles and several myelinated axons surrounded by Schwann cells (A). Collagen matrix in bioimplant rat (B) shows randomly dispersed collagen fibers and many myelinatcd axons surrounded by Schwann cells generally oriented along the long axis of the graft. A, X 15,000; B, X 15,000.

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FIG. 3. Catecholamine histofluomscence microscopy of proximal tissue-scar fiber junction in a control rat. No catecholamine-containing varicosities (CCV) were seen within the scar tissue (st) fibers. Note CCV from proximal tissue (PT) stop short of the st creating a free zone (white dots) void of CCVs. SPG method. X300. Bar = IO pm.

examined. Although the scar fibers and CM autofluoresce, it was easy to discern any CCVs within them because the CCVs were bright, round, and about 0.5 pm in diameter and their fluorescence was more intense than the pale, lime-color autofluorescence characteristic of collagen (Figs. 3 and 4). Autofluorescence due to tissue debris or blood elements was easily distinguished from tissue CCV. The former were seen as pale orange, irregularly shaped, and remained in the tissue when the section were steam-heated 2

FIG. 4. A-histotluorescence microscopy of proximal tissue (PT) collagen matrix (CM) junction in gratted rat. Note catecholamine-containing varicosities (CCVs) coursing from the PT into the CM and continuing up the CM (top). B-CCV bundles with long-axis orientation in midregion of the CM and at the distal tissue (DT)-CM junction. C-CCV fibers were seen at the distal junction and within distal tissue itself. The CCVs within distal tissue were smaller than within the CM or proximal tissue. D-blood vessels(BV) within the CM with adrenergic CCV (1) contacts on the media layer (M). These periarterial CCV contacts were more common in the smaller arterioles (bv). SPG method. X300. Bars = 10 pm.

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min or when glyoxylic acid was deleted from the SPG solution. This catecholamine specificity test was described elsewhere ( 14). Figure 4 shows group X (CM) at the proximal (A), center of matrix (B), and distal and tissue interface (C). Numerous CCVs were seen in all three regions with the greatest density at the proximal-CM interface. Arteriolar vessels with CCVs in the media layer are seen in D taken from the center of the CM. Three distinct CCV types were seen in group X. A “swollen” reactivetype CCV was generally found in the proximal tissue near the junction with the CM and within the CM itself; the second type CCV was normal in size characteristic of central gray matter and was scattered through the proximal and distal tissue and CM (Fig. 4B); the third type was found almost exclusively in the distal cord tissue near its junction with the CM (Fig. 4C). These CCVs were significantly smaller than the other two types and resembled the “ultrafine” CCVs seen in cerebral cortex or hippocampus (14). Neurophysiological

Evaluation

Local Spinal Cord Blood Flow (ISCBF). Figure 1 shows the mean 1SCBF in the rats with the collagen bioimplant (group X) in comparison with the 1SCBF in controls (group C). The mean flow in the CM bioimplants was 190% higher than its counterpart in control scar fiber tissue. The mean flow 2 mm from the CM in a proximodistal direction were 66 to 88% higher than in the control counterpart regions. At 5 mm from the CM, the flows were not significantly different in the bioimplant rats from control tissue. Somatosensory Evoked Potentials (SEPs). Each rat was tested for SEPs at 90 days after cord transection. Eight of 20 rats with the CM in group X showed early SEP waveforms whereas none of 12 rats in group C showed any SEP pattern (Fig. 5-2). The SEP waveforms were generally present on the left or right side of the cortex, although in one rat, early SEP peaks were recorded bilaterally. The normal SEPs for rats are shown in Fig. 5-l and 53. In group X, three peaks ranging in latency from 9 to 32 ms were recorded in six rats and two peaks with latencies of 12 and 38 ms were seen in two rats (see Fig. 5-4). Two rats each from groups X and C underwent retransection of the spinal cord two levels above the lesion immediately following the SEPs. Again SEPs were monitored using the same parameters as before. The SEP pattern previously recorded in these two CM-treated rats was abolished and the SEPs resembled those recorded in control rats with transections (see Fig. 5-2). Neurological Examination. No rat in either group had rear-limb walking ability 90 days posttransection (dpt). At 90 dpt, sensory discrimination to pin prick appeared absent in both groups as were normal reflexes below the lesion. Reflex functions were tested

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FIG. 5. Femoral nerve (saphenous branch) somatosensory evoked potentials (SEP) of control (5-l) and treated rat (5-3) before injury and total spinal cord transection; 90 days after transection, no SEP waveforms were seen in controls (5-2). Early waveform SEP peaks ranging from 9 to 32 m/s were seen in 8 of 20 collagen matrix-treated rats (5-4). Median nerve SEPs (not shown) above the lesion were used as controls and were present in all rats a&x 90 days. Early waveform SEP peaks were abolished a&r retransection of cord above the lesion. The SEP tracings were made from polaroid-oscilloscopic summated responses.

as follows: Flexion reflex was tested by picking up the rat and pinching its toes with forceps; the normal reflex is to move the foot away. Grasp reflex was tested by picking up the rat and touching the palm with a wire (31). Motor examination showed that 6 of 20 rats in group X, but none in group C, could support their bodies by standing on their rear limbs and lightly grasping the edge of a bowl with their forepaws. Four of those 6 rats also showed SEP waveforms. Gait was tested on an obstacle course where the rats had to “lift” or “drag” their rear limbs to overcome 2-in. Styrafoam ridges along their path. This test showed the 6 group X rats that could support their body using rear limb support, also could “lift,” apparently by spastic extension and flexion, one or both limbs to overcome the obstacles. No rat in group C had this ability. Group X rats had significantly less biceps femoris muscle wasting than group C when the muscles were palpated for tone and atrophy.

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Bladder control returned after a mean duration of 19 days following cord transection in group C and after 14 days in group X. Because of the high standard deviations in both groups, this difference was not significant. Leg ulcers were seen in 10 of 12 group C rats and in 3 of 20 group X rats. This difference is significant below the 0.01% confidence level using a chi-square test. The average loss of weight (60 g) that followed surgery was similar in both groups of rats. No weight gain difference was observed in either group during the go-day observation period. DISCUSSION As summarized in Table 1, a number of factors have been proposed to explain the lack of significant axonal regeneration after complete spinal cord transection (9). We define an unfavorable tissue milieu as one containing fluid-filled pools, necrotic tissue, vacuoles, toxic chemicals extruded from damaged cells or blood vessels, and reduced tissue perfusion, all or some of which may be present after severe cord trauma. Our results from this and previous experiments indicate that such an environment is not in itself sufficient to arrest the passage of central catecholaminergic axons in cord or brain tissue (8, 10). In regard to the physical barrier (foreign tissue, gliosis, blood clot, tissue membrane, scar), we conclude that only collagen scar fibers pose a critical obstacle in central neuritic regeneration. We agree with Kiernan’s (21) statement that regenerating axons are never seen crossing dense collagenous scars. The scar tissue barrier could be both physical and chemical as catecholamine@ axons in group C (control) appeared to stop short of the scar fibers (Fig. 3) as though deflected by an “inhibitory substance” of unknown composition. That substance, possibly secreted from the scar fibers, may check the growth of regenerating axons before they reach the cicatrix (10). In further support is the observation that neurites present in silver stain preparations from the proximal cord tissue in the controls did not enter the scar fibers, but did so in CM-treated rats. In addition, a “free zone” 200 to 400 pm wide was seen at the proximal tissue-scar fiber junction where no CCVs were found (Fig. 3, white dots). These CCVs appeared deflected in a parallel direction before reaching the scar tissue. Although lack of axonal guidance through loss of trophic factors or an autoimmune reaction may not favor the axotomized regenerating processes, our results suggest that these two factors are not critical. Rather, the reversal of avascular or ischemic tissue appears highly crucial for successful axonal regeneration. For example, the poor fibroblastic proliferation and lack of catecholaminergic axons observed in the proximal tissue-scar fiber junction of control rats was also accompanied by significant reductions of spinal cord blood flow and vascular density compared with CM grafted rats (Fig. 1). In

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support of the need for axons to grow only in adequately vascularized tissue, Heinicke ( 19) reported that homotransplanted fragments of tendon will grow well in brain but that axons will regenerate only into the vascularized but not the avascular tendon grafts. In addition, Goldsmith el al. (16) showed that increasing spinal cord blood flow using omental transposition, can reverse motor deficits in spinal cord-injured dogs. The “XYZ” factor refers to “yet undiscovered” obstacle (s) that may prevent axonal regeneration and functional synaptic reconnections with central nervous system. Only further studies can indicate whether such a factor is involved in regeneration, and if so, to what extent. Such a factor may make it impossible at present to engineer functional sensory-motor axonal reconstruction in the transected spinal cord in spite of our awareness of other problem factors. Reports by several investigators have shown that tissue bridges inserted between transected spinal cord stumps can sometimes support axonal growth. These tissue bridges have been composed of muscle (1, 2) fetal cells (23, 24), peripheral nerve (29), or peripheral nerve bypasses (5). Our experimental model differs from other protocols on two essential points: (i) injury and stochastic approach, and (ii) use of a nontissue spinal cord bridge. The first point deals with methodology; that is, a selective component which is the model injury and a random component which is the testing variables. We believe that the common practice of studying spinal cord regeneration by transecting the cord and then examining the effects of a procedure or therapy has two inherent flaws: (a) First, the vast majority of clinical cord injuries are due to sudden impaction with compression of the tissue and not to transection of the spinal cord (9). (b) Second, the selection of a therapy or intervention soon after cord transection may provide better experimental results for the investigator, but is not clinically realistic. The reason is that no time is allowed for the development of tissue gliosis, hemorrhagic necrosis, gradual decrease in tissue blood flow, enzymic and transmitter changes within the cord tissue, and most important, the elaboration TABLE I Possible Factors Imoeding Regeneration in the Central Nervous System I. 2. 3. 4. 5. 6.

Unfavorable tissue milieu Physical (chemical?) barrier Lack of axonal guidance Autoimmune reaction Tissue ischemia “XYZ” factor

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of collagen scar fibers as occurs in subacute or chronic human injuries. These changes are not seen until many days after the spinal injury. We have attempted to minimize the effects of those two flaws by first subjecting the rats to a sudden impact injury known to produce permanent paralysis, then 10 days after this trauma, totally transecting the cord. This period allows the tissue to stabilize after the sudden trauma, and transecting the spinal cord many days later ensures that any neuritic growth that is recorded within the observation period is due to regeneration and not to “reactivation” of axonal function following concussion of the nerve fibers. In addition, delayed transection of the cord allows the removal of necrotic or avascular cord tissue by trimming the proximal-distal stumps. We believe this approach provides a more realistic human extrapolation if success is achieved with the experimental model. Considering the use of a CM bridge after complete transection of the cord, neurites were seen in continuity with the CM in proximal and distal cord tissue on silver-stained preparations and numerous catecholamine-containing varicosities were visible within the CM extending into the distal cord. If CCVs in the spinal cord gray matter are derived entirely from supraspinal sources, then transection of the cord should result in permanent loss of distal cord CCVs unless some regeneration of catecholaminergic axons has occurred. The absence of CCVs within the scar tissue fibers or distal cord tissue in any rat from group C (control) indicates central axonal regeneration in animals with a CM bridge. The possibility of catecholaminergic fibers in the CM and distal cord tissue of experimental rats arising from peripheral blood vessels or tissue is not supported by the morphologic findings. First, catecholaminergic fibers were not seen in the scar fibers or distal cord tissue of control rats even though the cord was damaged in the same fashion as in the experimental animals. Second, this phenomenon has not been documented in our spinal cord studies (IO, 11, 13) nor those of others (2, 24). Third, the CCVs seen in the experimental rats were oriented longitudinally, that is, parallel to the long axis of the spinal cord, rather than perpendicular to it as expected if the neurites were originating from perforating blood vessels. Fourth, the CCVs were not seen coursing along blood vessels either within the CM or in the distal cord tissue (see Fig. 4A-C). Finally, there was no morphologic evidence either by light or electron microscopy that revascularization of the CM or distal cord tissue was achieved by peripheral microvessels. The majority of new capillaries that anastomosed with larger arterioles in the CM had an endothelial cell layer bounded by a basal lamina and were indistinguishable from other microvessels in the central nervous system. The occasional presence of islands of unmyelinated and myelinated axons seen in control scar tissue suggests two possibilities. (i) The axons are growing from central cord tissue into the scar fiber region and continue along the

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length of the scar tissue. (ii) The axons are originating from the transected dorsal root ganglia. Because neurites were not seen within proximal and distal junction to the scar tissue using the Bodian silver stain and as no CCVs were evident within the scar fibers or its distal cord stump, the second possibility, that is, the axons seen with EM are from the sectioned dorsal root ganglia, seems more plausible. The presence of similar and more numerous axons in the CM suggests that dorsal root ganglionic axons enter the CM from the periphery in addition to the central axons extending into the CM from the proximal cord. The positive SEP findings in some CM-implanted rats also lend support to this possibility. The observed axonal association with Schwann cells within the central nervous system is a curious but previously described finding. Alter a compression lesion (17) or chemical axotomy of spinal cord fibers (3), the axonal regrowth in the tissue is accompanied by Schwann cells which migrate from nearby entry zones in the periphery in order to repair the damaged tissue. These Schwann cells, which are normally associated with peripheral axons, appear to form myelin for demyelinated central axons. Thus, central myelin and axonal repair does not appear to receive much help from oligodendrocytes, at least in those model injuries (3, 17). The presence of myelinated axons associated with Schwann cells in the damaged spinal cord indicates that the origin of the axons may be central, peripheral, or both and that further evidence is necessary to resolve this question. One of our preexperimental hypotheses was that neither axons nor fibroblasts will grow well within any intercalated bridge without an adequate blood supply. Preliminary experiments in rat brain showed that revascularization of damaged central nervous system tissue returned within 3 days when CM was used (8). Good vascular anastomoses between cord tissue and CM was demonstrated by histologic preparations as well as from the recording of local spinal cord blood flows (1SCBF). We showed elsewhere that normal rat SCBF varies from a high of 63 in gray matter to a low of 18 ml/100 g tissue/min in white matter ( 11, 18). The mean 1SCBF in the CM was 29 ml/ 100 g tissue/ min which is between normal mean white and gray matter flow values, but still 190% higher than scar tissue ISCBF. In addition, the 1SCBFs were significantly higher (66 to 88%) 2 mm in a proximodistal direction from the CM than in control scar tissue. The 1SCBF was the same in both groups of rats 5 mm from the CM or scar tissue, a finding that appears reasonable because the rat vascular supply to the cord is provided mainly by the ventral root arteries that feed each spinal segment (30). Consequently, the blood supply and flow at several levels above and below the cord lesion would not be affected by the induced trauma. An interesting finding in our study was the presence of adrenergic CCVs within the neovascular arterioles found within the CM. This perivascular

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innervation which was seen only in CM-treated rats, was identical to that reported in mammalian brain (6, 27). If this is confirmed, it would suggest a central neural control of spinal cord blood flow by adrenergic neurons. Evoked potentials indicated that 8 of 20 rats with the CM bridge had a return of the early SEP waveforms when tested 90 days alter spinal cord transection. Because the SEP response was absent in comparable control animals when nerves below the lesion were stimulated, sensory pathway reconstruction may have been achieved in some of the experimental rats receiving the CM. The extent and significance of this reconstruction is at present unknown, but on-going studies using tracer labeling techniques should clarify this question. The presence of early SEP waveforms in group X rats was not substantiated by sensory return in these animals as tested by pin-prick or pinch stimulation. This suggests that the return of SEPs after neuronal regeneration may precede gross sensory return as it does after acute, reversible, spinal cord injury (9, 25,28). The mechanism underlying this phenomenon is not clear, but if this is the case, SEPs could become an extremely important prognosticating tool in evaluating potential sensory return after permanent cord injury and chronic paralysis. Use of the collagen matrix has several appealing features. It can be dispensed using a prepackaged syringe containing the sterile semifluid at 4°C. As a semifluid, the CM will fill a space or a tissue defect by gravity; such as the gap created after ablation of spinal cord tissue, then harden at body temperature to create a tight junction with the tissue edges (8, 10). The matrix remains pliable and the collagen fibers remain loosely and randomly interwoven, providing support rather than a barrier to colonizing host cells such as fibroblasts, neurites, and to blood vessels. The nourishment provided by the capillary network which anastomoses with the cord may be one of the most redeeming qualities of the CM. There was no evidence of inflammatory or immunologic reaction to the CM by the host tissue, a problem that sometimes arises even in allogeneic tissue grafts. The tight physical continuity between the CM and the proximal and distal cord stumps after 90 days appeared to provide a good structural conduit of the collagen with the cord and may have facilitated the migration of central neurites, fibroblasts, and blood vessels into the matrix. Experiments using this CM in human epidermis have shown that several months after subcutaneous implantation, the collagen mass was accepted by the host tissue as a permanent connective tissue matrix (22). The major difference between the implanted CM and the collagen scar fibers elaborated by the host after injury, involves structural configuration of the fibers. The CM fibers were loosely meshed when examined by silver stains and EM; collagen scar fibers appeared structurally tighter and more oriented than CM fibers. These dif-

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ferences may result in a less densely bound matrix that supports the growth and colonization of cells from the host tissue as represented by the CM, or in a tightly arranged barrier as seen with the collagen scar. The scar fibers may prevent neuritic growth directly by their tight configuration, by their release of a chemically unknown substance that may prevent further neuritic growth, or, indirectly, by their decreased support of blood vessels (and consequently reduced nourishment), or by various combinations of all three mechanisms. The results of this and a previous study (IO, 12) show the feasibility and potential of the CM bridge in providing a relatively nonhostile bridge for blood vessels, fibroblasts, and neurites growing from the damaged host tissue. Although no rat in either group regained normal motor function, the results indicate that some behavioral, morphologic, and neurophysiologic differences exist after complete spinal cord transection between a CM bridge implant and apposition of the cord stumps. REFERENCES 1. BJERRE,B., A. BJ~RKLUND, AND U. STENEVI. 1973. Stimulation of growth of new axonal sprouts fmm lesioned monoamine neurons in adult rat brain by nerve growth factor. Bruin Res.,6th 161-176. 2. BJ~RKLUND, A., R. KATZMAN, U. STENEVI, AND K. A. WEST. 1971. Development and growth of axonal sprouts from noradrenaline and 5-hydroxytryptamine neurones in the rap spinal cord. Brain Res. 31: 21-33. 3. BLAKEMORE, W. F. 1975. Remyelination by Schwann cells of axons dcmyelinated by intraspinal injection of 6-aminonicotinamide in the rat. J. Neurocytol. 4: 745-757. 4. B~DIAN, D. 1937. The staining of nervous tissue with activated protorxol. The role of 6xatives. Anat. Rex 69: 153-162. 5. DAVID, S., XND A. J. AGUAYO. 1981. Axonal elongation into peripheral nervous sysem “bridges” alter central nervous system injury in adult rats. Science 214: 931-933. 6. DE LA TORR!Z,J. C. 1976. Evidence for central innervation of intracerebral blood vessels. Neuroscience 1: 455-457. 7. DE LA TOWE, J. C. 1980. An improved approach to histofluorescence using the SPG method for tissue monoamines. J. Neurosci. Meth. 3: l-5. 8. DE LA TORRE, J. C. 1981. Regeneration of catechohunine fibers into a collagen bioimplant after cortical ablation. Sot. Neurosci. Abstr. 7: 260. 9. DE LA TORRE, J. C. 1981. Spinal cord injury: review of basic and applied research. Spine 6: 315-335. 10. DE LA TORRE, J. C. 1981. Catecholamine fiber regeneration across a collagen bioimplant alter spinal cord transection. Brain Res. Bull. 9: 545-554. 11. DE LA TORRE, J. C., AND J. E. BOCGAN. 1980. Neurophysiological monitoring in rat spinal cord trauma. Exp. Neural. 70: 356-370. 12. DE LA TORRE, J. C., P. K. HILL, M. GONZALEZ-CARVAJAL, AND J. C. PARKER. 1982. Regeneration of transected spinal cord axons into a collagen bioimplant. Neurology 32: A213. 13. DE LA TORRE, J. C., AND M. GONZALEZ-CARVAJAL. 198 1. Steady state drug or fluid delivery to injured or transected spinal cord of rata. Lab. Anim. Sci. 31: 701-703.

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