Functional restoration of the traumatically injured spinal cord in cats by clonidine.

The long-term, chronic, paralysis resulting from spinal cord injury in the cat has been reversed by the use of an alpha 2-adrenergic receptor agonist, clonidine. Administration of this drug resulted in "normalization" of sensory-motor and autonomic dysfunctions. Preliminary studies of the clonidine in humans with traumatically injured spinal cord indicate that autonomic dysreflexia can be controlled and spasticity minimized. The data suggest that biochemical and pharmacologic manipulation of receptors may ameliorate paralysis following traumatic injury to the spinal cord as well as to the brain and brainstem.

Experimental alterations in cyclic adenosine monophosphate concentrations in the cat basilar artery.

Levels of cyclic adenosine monophosphate (AMP) in the basilar artery and in circulating blood of cats were determined after the production of spasm by topical application of blood to the vessel and following treatment with agents known to alter cyclic AMP. Isoproterenol, known to stimulate adenyl cyclase, and aminophylline, a phosphodiesterase inhibitor, were studied alone and in combination. Cyclic AMP of the basilar artery fell from a mean control value of 43 to 26 pmoles per milligram of protein following the production of vasospasm. Intravenous administration of isoproterenol alone and in combination with aminophylline produced dilatation of the basilar artery, which was associated with a marked rise in the cyclic AMP concentration in the vessel. The finding that cerebral vasospasm is associated with a fall and vasodilation with a rise in cyclic AMP concentration supports the hypothesis of an active role for cyclic nucleotides in the regulation of cerebrovascular smooth muscle tone.

A modified peroxidase--antiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord.

A procedure is presented which modifies the Sternberger peroxidase--antiperoxidase (PAP) technique in order to visualize additional amounts of immunodeposits representing the antigen substance (SP) in 5-micrometer paraffin tissue sections of rat spinal cord. For increased sensitivity, the new procedure utilizes a "double bridge" and diaminobenzidine in low pH buffer. The modifications have made possible the visualization of immunoreactive beaded processes and punctate bodies, which were then traced to determine patterns of SP circuitry. Using the modified PAP procedure, the greatest number of immunoreactive processes appeared in the dorsal horn, where some punctate bodies and varicose processes could be seen adjacent to the myelinated afferent fiber bundles that penetrate the substantia gelatinosa as dorsal root collaterals. Additional immunoreactive processes and punctate bodies coursed through the myelinated afferent fiber bundles that penetrate the dorsolateral white matter, and extend into the intermediolateral gray region. Substance P was also identified within immunoreactive processes found in Rexed's laminae V and VI, as well as the central canal region, the dorsal gray commissure, and the ventral gray and white commissures. Since the modifications improved the visualization of SP-containing processes in sparsely populated regions of the spinal cord, especially the ventral horn, they may be useful in demonstrating other antigens that normally occur in small quantities within tissues.

Prevention of damage in acute spinal cord injury by peptides and pharmacologic agents.

Cats were used as models of traumatic spinal cord injury. Each experimental animal received a 500 g-cm force to the exposed dura at the level of thoracic fourth vertebra. Somatosensory evoked potentials (SEPs), carotid arterial blood pressure (BP), and abdominal aorta blood flow in the treated groups were compared with those of the control group. The three treated groups received naloxone (5 mg/kg), TRH (5 mg/kg), and a combination of methyl-prednisolone sodium succinate (MP, 35 mg/kg) and epsilon-aminocaproic acid (EACA, 350 mg/kg). The SEPs which were done only in the naloxone treated group approached "normalcy" 24-26 hours after trauma as compared with the absence of SEPs in traumatized untreated group. In all three groups, the treatment increased the blood flow in abdominal aorta significantly. Morphine sulfate increased substance P (SP) immunoreactivity in the dorsal and ventral gray matter. Naloxone not only reversed this effect, it depleted SP below the saline control level. In order to establish that lipid free radicals are responsible for damage to biological membranes, their effects were also investigated in vitro: 14C-GABA uptake by mouse cortical slices which had decreased by 33% in the presence of superoxide (. O-2) generating system, horseradish peroxidase (HRP), was reduced only by 9% when superoxide dismutase was added to the medium. The latter also protected the nerve endings from damage by (. O-2) as examined by electron microscopy. It is concluded that the agents used in this study produce their ameliorating effects by virtue of their anti-inflammatory, anti-oxidant, and membrane stabilizing properties in addition to their effect on enhancing the regional microcirculation. The release of SP by naloxone may be responsible for the increase in blood flow. The consequences of traumatic injury as depicted in Fig. 1 are discussed at length.

Alterations of neuroendocrine functions in spinal cord injury.

A transverse myelopathy causes a marked dysfunction in the negative feedback of some of the hypothalamo-hypophyseal-target organ axes. Spinal cord injured (SCI) humans with lesions above T6 exhibit significantly lower serum LH and testosterone but not FSH. The parathyroid hormone-thyrocalcitonin regulation of bone metabolism is disturbed; the higher the lesion, the greater is the loss of mineral and matrix components of the bone. Further, the response to insulin induced hypoglycemia is blunted in SCI with high lesions, and their basal levels of norepinephrine, epinephrine, and cortisol are significantly lower than those of controls. Distension of the bladder or rectum, however, produces marked release of norepinephrine and concomitant increase in sympathetic activity. The results suggest impaired hypothalamo-hypophyseal-testicular, -adrenocortical, - adrenomedulary, -sympathetic axis dysfunction.

Substance P and leucine-enkephalin changes after chordotomy and morphine treatment.

Spinal cords of rats, cats and monkeys were transected; the animals were perfused at varying times. Other rats were injected with morphine and perfused 10 days later. Immunocytochemistry shows substance P (SP) present in control animals primarily in the substantia gelatinosa (SG) of the dorsal horn of the spinal cord. Slight SP immunoreactivity is found in the ventral horn and near the central canal. Starting a few days after transection, there is a buildup of reaction product in the dorsal horn, in sections cut from below the lesion; staining above remains the same. With time, after chordotomy, SP immunoreactivity appears in fibers in lamina V, only in sections below the lesion. Leu-enkephalin (LE) is also found in the SG, however, it is also present in quantity in the ventral horn and central canal areas. Chordotomy has no effect on its distribution indicating LE is intrinsic in the cord and probably contained within interneurons. Morphine increases SP immunoreactivity in the SG, laminae I, IV and V, and in the ventral horn, suggesting morphine analgesia is due to inhibition of intraneuronal SP release in regions specifically associated with pain--SG and lamina V.

Alterations of immunoreactive substance P and enkephalins in rat spinal cord after electroacupuncture.

A form of electrically-induced analgesia known as electroacupuncture was administered to rats bilaterally at the point "Huan-tiao." Compared with untreated rats, treated rats showed altered pain thresholds characterized as low, intermediate, and high. From immunocytochemical studies, the spinal cords taken from the treated rats exhibited differences in immunoreactivity for substance P (SP), methionine- and leucine-enkephalins (ME and LE respectively). By densitometry, the altered levels of immunoreactive (IR) peptides correlated with the pain thresholds in specific ways. That is, high pain threshold correlated with the visualization of increased IR-SP adn IR-LE within neuronal processes throughout the dorsal horn substantia gelatinosa. In the same specimens, decreased IR-ME could be seen. In contrast, low pain threshold correlated with decreased IR-SP and IR-ME. IR-LE showed a concomitant decrease in the medial substantia gelatinosa region, and slight, insignificant changes laterally. The data suggest that different degrees of analgesia induced by electroacupuncture result from the variable release of SP, ME, and LE in spinal regions associated with nociception. In terms of current models of pain processing, the data do not entirely support an axo-axonic interaction between enkephalin interneurons and SP terminals. Some modifications and an alternative model are considered.

Presence of leucine-enkephalin in organotypic explants of fetal mouse spinal cord.

Spinal cord explants with attached dorsal root ganglia (DRGs), from 14-day fetal mice were fixed at 1-3 weeks in vitro and incubated for leucine-enkephalin (LE) immunoreactivity by the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Results show long processes with labeled varicosities seen more often in dorsal regions of the cord explants. Stained punctate bodies and varicosities were often seen close to large cells in these cultures, whereas no label was detected in neuronal perikarya. A prominent laminar array of stained punctate bodies was noted in one cord explant, concentric with the perimeter of the explant. No LE label was detected in the neuritic outgrowths from the cord-DRG explants, whereas high levels of opiate receptors develop in these outgrowths, primarily on the DRG neurites, by 1-2 weeks in culture. The results indicate the presence of LE in explants of fetal mouse spinal cord with attached DRGs and offer an in vitro model system in which the onset and development of peptidergic neurons can be studied as they form functional cellular interrelationships with neurons bearing opioid and monoaminergic receptors in these organotypic cultures.

Treatment of mammalian spinal cord injury with antioxidants.

After spinal cord injury, two groups of cats were treated with a combination of methylprednisolone sodium succinate (MP, 35 mg/kg) and epsilon-aminocaproic acid (EACA, 350 mg/kg), and guanabenz acetate (0.65 mg/kg). Guanabenz acetate was administered twice daily for 8 weeks. In the first group, the treatment significantly increased blood flow in the abdominal aorta. All cats treated with guanabenz acetate 3 hr after spinal cord contusion had return of micturition and none suffered complete paraplegia. Four animals had partial and the other four had complete motor recovery. A superoxide (O2-.) generating system, horseradish peroxidase, decreased [14C]gamma amino butyric acid uptake by mouse cortical slices by 33% but when superoxide dismutase was added to the medium, the uptake was reduced by only 9%. The nerve endings were also protected by superoxide dismutase from morphologic damage by O2-. as observed by electron microscopy. The agents used in these studies produce their ameliorating effects by virtue of their anti-inflammatory, antioxidant, and membrane stabilizing properties, and enhancing the regional microcirculation. In addition to having these properties, guanabenz acetate is also an alpha 2 adrenoceptor agonist.

Correlation of changes in the GABA-ergic system with the development of spasticity in paraplegic cats.

Following spinal cord transection there occurred decreases in Km and Vmax of glutamate decarboxylase (GAD) both above and below the lesion, and an initial decrease in the concentration of GABA. Concomitantly, there was a gradual decrease in presynaptic inhibition. Eight to 12 weeks after spinal cord transection, Km and Vmax for GAD returned to control values, but the GABA content of the spinal cord below the lesion increased significantly and presynaptic inhibition became maximally depressed. These results suggested that during the chronic phase of spinal cord injury there is a decrease in release of GABA, the interneuronal inhibitory neurotransmitter which mediates presynaptic inhibition. Diazepam, a GABA enhancer, increased presynaptic inhibition in acute and chronic spinal cats, this being accompanied by a reduction in somatic muscular spasticity. The degree of this enhancement by diazepam, however, is attenuated with gradual loss of presynaptic inhibition. In the acute cat, a conditioning volley applied to cutaneous afferents blocked the inhibition of the monosynaptic response to extensor motoneurones. In contrast, in chronic spinal cats (eight to 12 weeks), the duration of complete blockade was markedly reduced and was followed by a prolonged period which cutaneous nerve stimulation potentiated the monosynaptic discharge. Similar to GABA, there also occurred an increase of substance P below the level of the lesion. Other neurotransmitters (e.g., norepinephrine, serotonin) accumulated above and disappeared below the transection level. Although somatic msucular spasticity appears to be, to some extent, due to GABA dysfunction in the spinal cord, alterations in "normal" functioning of other neurotransmitters and the loss of supraspinal control also contribute to this state.

Autonomic dysreflexia: pharmacological management of hypertensive crises in spinal cord injured patients.

Interruption of autonomic pathways by spinal cord injury (SCI) causes dysfunctional autonomic dysreflexia (AD), which was first described in 1917, still remains unrecognized by those in the medical profession not involved in SCI care. Autonomic dysreflexia is a syndrome generally manifest by cardiovascular symptoms and characterized by paroxysmal hypertension. These symptoms appear in patients with spinal cord injury above the sympathetic outflow from the spinal cord (T6). Since patients with high level SCI are usually hypotensive, the high blood pressures that develop during AD represent pressure changes of a magnitude that can cause cerebrovascular accidents and death of the subject. We discuss the therapeutic interventions that abate and curtail the symptoms and prevent the catastrophic sequelae of autonomic dysreflexia.