Hindlimb paralytic effects of arginine vasopressin and related peptides following spinal subarachnoid injection in the rat.

Intrathecal (IT) injection of arginine vasopressin (AVP) in rats caused a transient (less than 30 min), dose-related paralysis of the hindlimbs, loss of hindlimb and tail nociceptive responsiveness, and increased mean arterial pressure. Motor dysfunction was produced with comparable potency by lysine vasopressin (LVP) and arginine vasotocin (AVT); oxytocin (OXY) was approximately 1000 times less potent. Paralysis induced by these peptides was selectively blocked following IT pretreatment with 0.5 nmoles of the vasopressin V1 receptor antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine] Arg8-vasopressin (d(CH2)5[Tyr(Me2)]AVP). Pressor and antinociceptive responses to AVP were also blocked by this compound. However, at higher doses (2-5 nmoles, IT), d(CH2)5[Tyr(Me2)]AVP produced hindlimb paralysis, antinociception, and pressor responses by itself. In contrast to the fiber degeneration, cell loss, and necrosis found in lumbosacral cords of rats persistently paralyzed by other peptides (dynorphin A, somatostatin, and ICI 174864), neuropathological changes were not evident in spinal cords of rats transiently paralyzed by IT AVP. These results indicate that AVP-related peptides affected diverse spinal cord functions through interactions with a V1-like receptor. The similar pattern of cardiovascular and antinociceptive responses to other peptides (dynorphin A, somatostatin, and ICI 174864), which also caused hindlimb paralysis, suggests that the former responses may actually reflect the nonselective consequences of a peptide-induced disruption of spinal cord function, rather than specific shared pharmacological effects.

Cultures enriched in Schwann-like cells from dissociated nerve segments of the adult cat.

Methods for producing Schwann cell-rich cultures from nerve segments of the adult cat were investigated. Efficient dissociation was achieved with a combination of collagenase and trypsin. Differential attachment of elements within the resulting suspension allowed production of Schwann cell-rich cultures derived by outgrowth from slowly attaching segments of degenerating myelin sheath tube containing Schwann-like cells. Rapid trypsinization during subculture further increased the proportion of Schwann-like cells in these cultures to 90% or more.

Dynorphin A-induced rat hindlimb paralysis and spinal cord injury are not altered by the kappa opioid antagonist nor-binaltorphimine.

The selective kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI) was used to distinguish a kappa opioid component in the mechanisms underlying the hindlimb paralysis, ischemia, and neuronal injury induced in the rat by the kappa opioid agonist dynorphin A. Spinal intrathecal (i.t.) injection of nor-BNI (20 nmol) either 15 min or immediately before i.t. injections of 5 or 20 nmol of dynorphin A failed to alter the dynorphin A-induced disruption of hindlimb motor function and nociceptive responsiveness. Nor-BNI also did not change the 3-fold increases in cerebrospinal fluid lactate concentrations produced by 20 nmol of dynorphin A. Neuroanatomical evaluations revealed that the cell loss, fiber degeneration, and central gray necrosis in lumbosacral spinal cords of rats treated with 20 nmol of dynorphin A were not altered by nor-BNI (20 nmol, i.t.). Thus, the spinal cord injury and associated neurological deficits resulting from i.t. injection of dynorphin A appear to be primarily, if not totally, attributable to its non-kappa opioid action(s).

Non-neuronal cell cultures from dorsal root ganglia of the adult cat: production of Schwann-like cell lines.

There are several methods available for the production of Schwann cell cultures from fetal and neonatal peripheral nervous tissue. We have investigated methods for producing Schwann cell-rich cultures from adult tissue. Dorsal root ganglia from normal adult cats were used to initiate explant cultures or subjected to primary dissociation. The resulting cultures were compared in terms of growth, the proportions of fibroblastic and Schwann-like cells in primary cultures and the effects of subculture on the relative frequency of these cell types. We found that excision and transfer of explanted ganglion pieces after 14 days in culture produced a secondary outgrowth rich in small, bipolar, spindle-shaped Schwann-like cells. Subculture of this outgrowth produced secondary cultures of predominantly Schwann-like cells with typical spindle-shaped morphology. The use of antimitotic agents in the media to inhibit fibroblast growth was not observed to be necessary or beneficial with this adult tissue. Primary dissociation of ganglia with enzymes (trypsin or collagenase) and mechanical agitation was even more effective in producing secondary cultures and cell lines that were, by morphological criteria, predominantly or exclusively Schwann-like cells. One of these Schwann-like cell lines, designated GSA, has been carried over 24 subcultures while retaining characteristic Schwann cell morphology. Cells of this line have been examined by scanning and transmission electron microscopy. Karyotype analysis indicates a chromosome complement consistent with the species of origin, a normal cat.

Somatostatin causes vasoconstriction, reduces blood flow and increases vascular permeability in the rat central nervous system.

Using radiolabeled microspheres, spinal cord blood flow was measured after spinal subarachnoid injections of 3.1- to 12.5-nmol doses of somatostatin through either indwelling i.t. catheters or acutely inserted intervertebral needles. With either injection technique, somatostatin caused significant dose-dependent reductions in thoracic and lumbosacral blood flow that could be partially blocked by a 5-min preinjection of the somatostatin receptor antagonist cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)], which has previously been shown to block the hindlimb flaccidity produced by these doses of somatostatin in conscious rats. The duration of these blood flow changes were appreciably less in the rats injected through indwelling i.t. catheters. Somatostatin-induced reductions in spinal cord perfusion were accompanied by transient pressor responses, reduced cardiac output, 3-fold increases in spinal cord cerebrospinal fluid lactic acid concentrations and breakdown of the blood-spinal cord barrier, as reflected by significantly increased extravasation of [125I]bovine serum albumin. By 24 hr postinjection, a 12.5-nmol dose of somatostatin caused appreciable spinal cord cellular injury, as evidenced by significant elevations in cerebrospinal fluid concentrations of lactate dehydrogenase. After topical application to exposed pial vessels of the parietal cortex, comparable doses of somatostatin caused immediate intense dose-related arteriolar vasospasm and subsequent extravasation of the visible macromolecular tracer Evans blue dye. We conclude that somatostatin has significant vasoconstrictory effects on the blood vessels of the brain and spinal cord of the rat that must be recognized and appreciated when studying its neuropharmacological actions in vivo.

Reconstruction of the contused cat spinal cord by the delayed nerve graft technique and cultured peripheral non-neuronal cells.

Previously, surgical reconstruction of the transected dog spinal cord by the delayed nerve graft technique has been shown to result in reinnervation of the nerve graft by axons. In the present study, we compared the results of surgical reconstruction of the severely contused cat spinal cord by the delayed nerve graft technique alone to those after reconstruction with a similar nerve graft plus cultured peripheral non-neuronal cells implanted between the grafted nerve and the spinal cord stumps. The spinal cord-nerve graft junction was examined by light and electron microscopy. The cultured cells were prelabelled with tritiated thymidine and their location after implantation determined by autoradiography. By 3 days after spinal cord reconstruction, the prelabelled cells were present at the junction and had migrated into the nerve graft and also into the spinal cord stumps where they were observed near axons. By 7 days, physical connections were observed bridging the junction between the spinal cord and nerve graft and axons ensheathed by Schwann cells had already penetrated at least 1 mm into the nerve graft. Wound healing took at least a week longer in animals repaired with a nerve graft alone. At one year or later after reconstructive surgery, in both groups of animals, the grafted nerve was reinnervated with myelinated and unmyelinated axons. Thus, the severely contused cat spinal cord could be reconstructed with the delayed nerve graft technique alone but the use of the cultured cells appeared to enhance wound healing and decrease the time required for axon elongation into the nerve graft.

Dynorphin A-induced rat spinal cord injury: evidence for excitatory amino acid involvement in a pharmacological model of ischemic spinal cord injury.

Dynorphin A reduced lumbosacral blood flow, elevated cerebrospinal fluid lactic acid concentrations and caused flaccid hindlimb paralysis and striking neuropathological changes after its injection into the spinal subarachnoid space in rats. Coadministration of the vasodilator hydralazine substantially eliminated the paralytic, anaerobic metabolic and neuropathological responses to dynorphin A. In contrast, in concentrations up to 1 mM, dynorphin A did not alter the viability of cultured rat spinal cord neurons. Thus, it appears that this peptide lacks direct neurotoxic effects and that neuronal injuries in vivo result primarily from ischemia associated with dynorphin A-induced blood flow reductions. NMDA receptor antagonists significantly improved recovery from dynorphin A-induced hindlimb paralysis, and substantially eliminated neuropathological changes without attenuating the acute blood flow reductions or lactic acid elevations. Additionally, glutamate and aspartate concentrations were increased significantly in spinal cord cerebrospinal fluid samples removed during the time that peptide-induced spinal cord blood flow reductions were observed. In contrast, neither amino acid concentration was elevated in media removed after 1-hr exposure of spinal cord neuronal cell cultures to 100 microM concentrations of dynorphin A. These results indicate that the paralysis and spinal cord injuries produced in rats after spinal subarachnoid injection of dynorphin A result predominantly from spinal cord ischemia, and further identify excitatory amino acids and N-methyl-D-aspartate receptor mechanisms as important mediators in this injury model.

Effects of NMDA receptor antagonists following spinal ischemia in the rabbit.

Evidence has accumulated to implicate the excitatory amino acid neurotransmitters, glutamate and aspartate, in the pathophysiology of central nervous system (CNS) ischemic injury. It appears from both in vivo and in vitro experiments that they exert their excitotoxic effects in CNS ischemia by their actions at the N-methyl-D-aspartate (NMDA) receptor complex. In the present study, we examined the effects of MK-801 and ketamine, two noncompetitive NMDA receptor antagonists, in a model of spinal cord ischemia in conscious rabbits produced by occluding the infrarenal aorta for 25 min. Five minutes after reperfusion, animals were treated with either saline, ketamine, or MK-801. By 6 h postreperfusion, all treatment groups exhibited an initial recovery of hindlimb motor function, after which the saline- and ketamine-treated groups had a similar progressive deterioration in function over the next 48 h. However, the MK-801-treated rabbits continued to recover motor function such that neurological scores in these rabbits were significantly improved relative to those of the saline-treated animals at 48 h. Histopathological evaluation showed that MK-801-treated rabbits tended to have a lesser degree of central gray matter necrosis. These results indicate that MK-801 protected against the secondary deterioration associated with this model and strengthen the potential therapeutic use of NMDA receptor antagonists in the treatment of CNS ischemia.