Recent Publications by CFE Educators

We used pharmacological and surgical methods to determine the contribution of several neural components to joint injury in rats with adjuvant-induced arthritis. Both neonatal administration of capsaicin, which eliminates small-diameter afferents, and peripheral sympathectomy, which depletes catecholamines, attenuated joint injury. In contrast, the arthritis was more severe in spontaneously hypertensive rats, which have increased sympathetic tone. To address the contribution of the central vs peripheral afferent terminal selectively, a group of rats underwent unilateral dorsal rhizotomy. These rats developed a more severe arthritis in the deafferented limb. The increase in arthritis severity produced by dorsal rhizotomy could be reduced by prior sympathectomy or, less effectively, by prior treatment with capsaicin. The latter observation suggests that large-diameter afferents that are cut during dorsal rhizotomy also influence inflammation. Finally, intracerebroventricular injection of morphine attenuated the severity of arthritis, possibly through activation of bulbospinal sympathoinhibitory circuits. Taken together, these data indicate that no one class of nerve fiber is wholly responsible for the neurogenic component of inflammation in experimental arthritis but that large- and small-diameter afferents, sympathetic efferents, and CNS circuits that modulate those fiber systems all influence the severity of joint injury in arthritic rats.

A novel therapy for rheumatoid arthritis, regional sympathetic blockade using guanethidine, was investigated in 24 patients with active disease. In a randomized double blind short-term (14 days) study, we evaluated the effect of therapy on subjective responses, change in pain, stiffness, and morning stiffness and no objective responses, change in pinch strength, grip strength, and joint tenderness. Compared to placebo, guanethidine produced a decrease in pain (p less than 0.025) and an increase in pinch strength (less than 0.025) over the 2-week duration of the study. The therapeutic effect of guanethidine may be mediated by an interruption of the proinflammatory effects of the sympathetic nervous system.

Two antigens, cholera toxin (CT) and a synthetic albumin-conjugated 16-residue peptide derived from the circumsporozoite (CS) protein of Plasmodium falciparum sporozoites, were tested as immunogens in rabbits. The malaria peptide-albumin conjugate by itself was completely nonimmunogenic, and although cholera toxin was immunogenic it also expressed considerable native toxicity. After attachment of CT to liposomes containing ganglioside GM1, toxicity of CT was completely eliminated and antigenicity was enhanced. Therefore liposomes may be capable of reducing toxicity of certain potentially dangerous antigens such as toxins. After incorporation of the malaria peptide-albumin conjugate into liposomes a high titre of specific antibodies was induced against the malaria peptide but not against albumin. These antibodies also reacted with native CS protein. Three adjuvants, including lipid A and two types of lipophilic muramyl dipeptide, were compared and found to be effective in liposomes. Based on the conversion of synthetic P. falciparum CS peptide from a nonimmunogenic to an immunogenic form and on the 'toxoiding' effect of liposomes for CT, it is concluded that liposomes should be considered as being a useful carrier for antigens and adjuvants for vaccines for poorly antigenic or toxic substances.

The projections of the nucleus raphe magnus (NRM) and the immediately adjacent reticular formation were studied in the macaque monkey following injections of the rostroventral medulla with 3H-leucine and examination of the resultant labeled axons and terminals by light and electron microscopic autoradiography. Five monkeys had accurately placed injections, which resulted in fiber pathway labeling that coursed caudally, laterally, and dorsally to project to laminae I, II, and V of subnucleus caudalis of the trigeminal and then traveled in the dorsolateral funiculus of the cord and terminated in similar laminae of the spinal dorsal horn at cervical levels. The pathway was only lightly labeled caudal to the cervical enlargement and could not be readily discerned above background in the thoracic or lumbar cord. Electron microscopy revealed that axons and terminals serving this system constitute a heterogeneous population. Large-diameter myelinated axons (3-6-micron diameter), small myelinated axons (0.75-3-micron diameter), and clusters of nonmyelinated axons were labeled. Terminals in laminae I, II, and V contained mixtures of clear round and granular vesicles or clear pleomorphic and granular vesicles or formed the central element in synaptic glomeruli. The labeled profiles formed asymmetrical or symmetrical synapses on medium and small dendrites; labeled axosomatic synapses were not observed. In rare instances there were contacts between labeled profiles and vesicle-containing structures, which were probably dendritic, but whether the NRM axon was pre- or postsynaptic to such structures could not be determined. It was concluded that the NRM in the monkey is organized in a manner quite similar to that previously described in the cat. The wide variety of fiber types and synaptic terminals serving this system suggests that different classes of neurons participate in it, probably using several transmitter substances that result in varying postsynaptic effects on neurons located in the trigeminal complex and dorsal horn.

We examined the differential effects of disease severity and pain on morbidity (as measured by weight loss and decrease in activity) in rats with adjuvant-induced arthritis. We found that eliminating nociceptive messages from affected extremities, although not significantly affecting the course of the disease, attenuated the morbidity observed in arthritic rats. When pain was factored out, the severity of the arthritis had no significant effect on the same measures of morbidity. These findings suggest that treatment of pain may reduce morbidity, and thereby may have significant benefits beyond that provided by slowing of the disease process.

Although very sensitive chromogens have been adapted for localization of horseradish peroxidase in anterograde and retrograde tracing studies, they have not been successfully applied in immunocytochemical studies. This report describes a protocol which uses benzidine dihydrochloride (BDHC) as the chromogen for light (LM) and electron microscopic (EM) immunocytochemical studies. The protocol is comparable to that used for tetramethylbenzidine, except that the pH of the reaction is above 6.0. At the LM level, the BDHC reaction product is bluish-green and crystalline. Both the color and form of the product are readily distinguished from the reddish-brown DAB reaction product. LM double-labeling studies are therefore feasible. The use of BDHC also increases significantly the sensitivity of the immunoreaction. Higher fixative concentrations can be used, less detergent is necessary, and higher primary antibody dilutions are possible. By osmicating at 45 degrees C in an s-collidine buffer it is possible to preserve the soluble BDHC reaction product for EM analysis. Immunoreactive cells are particularly well labeled with this new protocol. The BDHC crystals are easily detected at the EM level and can be distinguished from flocculent DAB reaction product. This feature makes EM double-labeling studies possible.

Although gold is one of the few therapeutic agents that has been proven effective in producing remission in patients with rheumatoid arthritis, its mechanism of action is unknown. Since nociceptive afferent and sympathetic efferent fibers of the peripheral nervous system contribute to the pathophysiology of inflammation, and since a known side effect of gold therapy is a polyneuropathy, we tested the hypothesis that gold is toxic to small-diameter peripheral nerve fibers in the rat. We found that prolonged treatment with gold, at the same dosage reported to be effective against adjuvant-induced arthritis in the rat, produced a significant decrease in the numbers of unmyelinated, but not of myelinated, axons. Gold treatment also elevated nociceptive thresholds in both articular and nonarticular structures. These results suggest that gold produces an antiinflammatory effect on arthritis by a neurotoxic effect on the peripheral nerves involved in neurogenic inflammation.

This study used antisera directed against glutamic acid decarboxylase (GAD), the biosynthetic enzyme for gamma-aminobutyric acid (GABA), to examine the light- and electron-microscopic distribution of presumed GABA-ergic synapses in the medullary homologue of the cat spinal dorsal horn, the trigeminal nucleus caudalis. At the light-microscopic level, immunoreactive terminals were concentrated in the superficial dorsal horn, laminae I and II. Colchicine was generally ineffective in revealing the distribution of cell bodies. However, in two successful cases, the majority of labeled cells were found in the magnocellular layer, ventral to the substantia gelatinosa, a region that had a lower density of immunoreactive terminals. Other labeled neurons were scattered in laminae I and II. A variety of synaptic arrangements were found at the electron-microscopic level. These derived from two types of labeled terminals. One contained both small round vesicles and large dense-cored vesicles. The second contained small round and pleomorphic vesicles. Some immunoreactive GAD terminals contained a few flat vesicles. Labeled terminals predominantly formed axodendritic synapses, via symmetrical contacts. Several axoaxonic arrangements were also observed. In most cases, the GAD terminal (which did not contain dense-cored vesicles) was presynaptic to another vesicle-containing profile, including the scalloped central terminal thought to derive from primary afferents. Another population of labeled GAD terminals was found postsynaptic to unlabeled vesicle-containing profiles, including central terminals. These data indicate that inhibitory GABA-ergic controls in the trigeminal nucleus caudalis involve both presynaptic and postsynaptic mechanisms and are probably mediated via direct contacts onto ascending projection neurons, as well as via synaptic contacts onto nociceptive primary afferent fibers. The transmission of nociceptive messages by neurons of the spinal cord dorsal horn and trigeminal nucleus caudalis is subject to a variety of segmental and supraspinal controls. Pharmacological and electrophysiological studies have implicated the biogenic amines serotonin and norepinephrine, and the endogenous opioid peptides enkephalin and dynorphin, in those controls (Basbaum and Fields, 1978, 1984; Basbaum et al., 1983; Basbaum, 1985).(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical analysis of the distribution of dynorphin B terminals in the sacral spinal cord of the cat revealed a pattern of staining very similar to that produced with antisera directed against the primary afferent derived, putative neurotransmitter, vasoactive intestinal polypeptide. Labeled axons and terminals were concentrated in lamina I and V and there was dense fiber staining in the tract of Lissauer. Of particular interest was the presence of immunoreactive axons in attached dorsal rootlets. To specifically focus on the possibility that some of the sacral primary afferent fibers are dynorphin-immunoreactive, we first tried to increase perikaryal labeling in the sacral dorsal root ganglia by topical treatment with colchicine. This did not produce immunoreactive labeling of cell bodies in the ganglia. Unilateral multiple dorsal rhizotomy (L5 to coccygeal 1), however, significantly decreased the staining of dynorphin-immunoreactive axons and terminals in the tract of Lissauer and in the dorsal horn of sacral segments ipsilateral to the deafferentation. No changes were detected in the lumbar cord. Finally, radioimmunoassay of caudal lumbar and sacral dorsal root ganglia was performed. Measurable immunoreactivity was found in all ganglia assayed, but, consistent with the histochemical analysis, sacral ganglia contained the highest concentration of immunoreactive dynorphin B. These data indicate that a significant component of the sacral spinal cord dynorphin terminal immunoreactivity derives from primary afferent fibers.(ABSTRACT TRUNCATED AT 250 WORDS)