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After complete cat spinal cord transection, a collagen matrix was used to bridge the gap. Vascular supply was increased to the transection site with an omental pedicle. Before hardening, either 4-aminopyridine, laminin, glia maturation factor, or lipid angiogenic factor were mixed into the collagen. Surgically reconstructed animals were compared to transection-only controls and observed for 90 days. Fluoro-Gold was injected distal to the transection site on day 75. Immunocytochemical examination of brain and spinal cord tissue was done on day 90. Examination revealed supraspinal catecholaminergic fibers present in the collagen bridge and distal cord tissue only in cats with surgical reconstruction. Fluoro-Gold particles were found localized in locus coeruleus and other noradrenergic pontine neurons. Distal to the transection, double immunostaining with synaptophysin and tyrosine hydroxylase or dopamine-beta-hydroxylase revealed dot-like deposits closely apposed to preganglionic sympathetic neurons suggestive of synaptic connectivity to these targets. Results indicate that considerable outgrowth of specific supraspinal fibers can be induced following spinal transection and reconstruction, and that such fibers may be extending and contacting appropriate distal target tissue in the cord.

Following complete transection of the spinal cord, cats were separated into 2 groups to undergo: (i) surgical reconstruction of the disconnected cord using a neuroactive agent mixed into a collagen matrix bridge and omental transposition and (ii) cord transection-only. After 90 days, animals were killed and the brain and spinal cord were removed for immunohistochemistry. Two weeks prior to sacrifice, spinal cord blood flows were measured and the retrograde axonal tracer Fluoro-Gold was injected below the transection site. Gross inspection of the spinal cords at autopsy showed excellent integration and continuity of the collagen matrix bridge with the proximal-distal stumps in the surgical reconstruction group. In the transection-only group, the proximal-distal stumps were connected by a fibrotic, often tapered in the middle, tissue bridge. Results show that omental transposition in the surgical reconstruction group increased spinal cord blood flow by 58% when compared to transection-only animals. Fluoro-Gold was found in mesencephalic and brainstem catecholaminergic and cholinergic neurons known to send axons to the spinal cord. Immunohistochemical staining with antibodies against catecholamine synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) showed that surgical reconstruction treated cat cords but not transection-only, developed dense bundles of dopaminergic and noradrenergic fibers which were present in the collagen matrix bridge and in the distal spinal cord. Extension of these catecholaminergic fibers in surgical reconstruction treated cats showed maximal outgrowth of 90 mm below the transection site when the neuroactive agent 4-aminopyridine was mixed into the collagen matrix. In addition, the synaptogenic marker synaptophysin (SYN) was observed on preganglionic sympathetic neurons in association with dopaminergic- and noradrenergic-containing varicosities distal to the collagen matrix bridge, an indication that neo-synaptic contacts may have been made on these previously denervated neurons. No TH, DBH or SYN was observed below the transection site in transection-only cats. These findings indicate that surgical reconstruction treated cords can develop dense supraspinal fiber outgrowth across a treated collagen matrix bridge fed by an omental blood supply and that these fibers may have made neo-synaptic contacts with appropriate distal spinal cord target tissue.

Spinal cord transection was induced in 3 groups of cats. The gap was surgically reconstructed using a collagen matrix bridge (Group COL), collagen matrix + pedicled omentum graft (Group COM), or gelfoam (Group GEF). After a variable observation period, animals underwent distal cord horse-radish peroxidase (HRP) injections, somatosensory evoked potentials recordings and polarographic measurement of local spinal cord blood flow (1SCBF) using the hydrogen clearance technique. The cord tissue was removed for histologic and immunohistochemical analysis. Results showed retrograde HRP labelling of proximal segmental cord neurons and somatosensory evoked potentials were present in group COM but not in COL or GEF treated animals. Local SCBF was 66% and 87% higher in COM than COL or GEF animals respectively but this increase could be reversed if flow from the pedicled omentum was clamped-off. Histologic examination of cord tissue after 45 days revealed the presence of catecholaminergic axons distal to the transection site in COM but not COL or GEF groups. Moreover, after 90 days, the rate and density of tyrosine hydroxylase immunoreactive (TH-IR) axons was 10-fold higher in COM than COL group and this was accompanied by a proportionate increase in the vascular density between the two groups. GEF treated animals showed no regeneration of transected fibers and poor blood flow pattern. These findings indicate that the placement of a pedicled omentum on a collagen matrix bridge results in near restoration of normal SCBF to the reconstructed cord region and is associated with marked regeneration of axons below the lesion site.

Rat spinal cords were subjected to a 200 g/cm force acceleration injury at T10. Ten days later, the cords were totally transected at T10 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 were examined for local spinal cord blood flows, 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 vessels to 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.

It is not known whether increasing the amount of blood flow to axotomized fibers in mammalian CNS can result in more robust sprouting. To find out, an intact pedicled omentum was surgically transposed to cover a collagen matrix gel used to bridge the transected cat spinal cord stumps. Control animals were similarly treated but did not receive the pedicled omentum. Twelve weeks after cord transection, animals receiving the pedicled omentum showed a 66% spinal cord blood flow increase over animals that did not. Moreover, treatment with the pedicled omentum increased the density of regenerating adrenergic axons 10-fold over the control group. These findings indicate that boosting flow with an omental graft to the collagen bridge site results in robust axonal outgrowth of spinal transected nerve fibers.

Adult rat spinal cords were perfused intracardially with aldehyde fixatives containing either sodium cacodylate or sodium phosphate buffer for EM studies. The perfusion flow rate of 24 cc/min for the first 3 min and 33 cc/min for completion of prefixation was found to be optimal. Ultrastructural examination of the L-1 fasciculus gracili, dorsal and ventral horns demonstrated that the quality of fixation is similar despite a two- to three-fold greater blood flow rate to the dorsal and ventral horns, respectively. Sodium cacodylate buffered perfusate provides superior clarity of organelle membranes, myelin lamellae and axoplasmic constituents when compared with sodium phosphate buffer.

A 10% calcium chloride solution or normal physiological saline was instilled onto the intact dorsal surface of the spinal cord via a specially constructed catheter-canopy system fixed above the T9 level in conscious rats. Within 5 min after calcium instillation rats developed flaccid paralysis of the lower limbs with sensory loss. Sensory loss was accompanied by abnormal or negative evoked potentials. Rats instilled with physiological or 10% sodium chloride remained normal. Rats were sacrificed at 1, 16 and 48 h post-calcium exposure and following full functional recovery from paralysis. Spinal cords were removed for histologic and high-performance liquid chromatography (HPLC) analysis. Histologic examination for catecholamines using SPG histofluorescence showed loss of catecholamine-containing varicosities in gray matter below calcium exposure which returned to normal levels upon sensorimotor recovery of hindlimbs about 14 days pce. Light microscopic examination of vascular permeability and general morphology of cord tissue axons and neurons remained normal in calcium and saline instilled rats. HPLC analysis of spinal cord below calcium exposure, also showed norepinephrine (NE) and 3-methoxy-4-hydroxyphenylglycol (MHPG) tissue level reductions which returned to normal upon sensorimotor recovery of paralysis about 2 weeks later. No significant changes were noted in dopamine or serotonin levels in any group. Our findings suggest an impairment of ascending and descending tract transmitter transport, specifically reflected in the noradrenergic bulbospinal pathway. The results implicate a neurofilament-microtubule disassembly in axonal cytoskeleton triggered by the sudden calcium influx.

A cell-free bovine derived collagen matrix was used to study potential axonal regeneration in transected rat spinal cord. Rats were initially subjected to a 200 g/cm force acceleration injury at T10 and 10 days later, the spinal cord was totally transected at the injury site. Controls had their spinal cord stumps juxtaposed end-to-end following transection. Experimental rats had 3-4 mm of spinal cord tissue trimmed from the proximo-distal stumps. The semi-fluid collagen material was implanted to bridge the proximo-distal ends and after several hours, the collagen graft polymerized to a firm gel. Rats were observed for 90 days. After 90 days, animals were evaluated using somatosensory evoked potentials, local spinal cord blood flow, and catecholamine histofluorescence in and around the site of transection. Results suggest that the collagen bioimplant can support the development of anastomotic blood vessels with the cord as well as provide a non-hostile environment to regenerating spinal cord axons.