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This study characterized by light and electron microscopy 49 photochemically induced lesions in adult rat spinal cord at 16 time intervals from 2 days to 17 months after lesioning. Vascular thrombosis, resulting from an intravascular photochemical reaction induced by a rose bengal/laser beam interaction, led within a few days to an extensive area of tissue deterioration. This area, termed the "lesion cavity" in contrast to the "secondary cavity" observed later, was at least 6 mm long and, at the epicenter, extended across most of the spinal cord width and from the dorsal surface to a level near the central canal. The area of spared tissue, 43% of the spinal cord cross-section at 2 days, did not change significantly between 2 and 56 days. Large numbers of macrophages populated the degenerating area by 5 days. This necrotic area was surrounded by a thin peripheral rim of largely intact white matter dorsally and laterally except at the epicenter where the white matter degenerated dorsomedially. In these peripheral regions, demyelination and, by 14 days, remyelination by both oligodendrocytes and Schwann cells (SCs) were evident. By 28 days, far more SCs (and meningeal cells) had entered the dorsal spinal cord, typically at the epicenter where meningeal thickening was most striking, and had migrated farther into the lesion cavity. These SCs and the axons they myelinated remained prominent in dorsal regions for many months, particularly at the epicenter; the proportion of SC to oligodendrocyte myelin diminished away from the epicenter. By 8 weeks, the lesion cavity was considerably diminished in size and thereafter it contained scattered macrophages, SC-myelinated axons, and blood vessels, primarily medially owing to flattening into clefts bilaterally. The cavity was partly bordered by astrocytes whose surfaces toward the lesion cavity were highly irregular and coated with basal lamina. Bare axons, consistently seen by electron microscopy at 5 days to 6 months, were typically ensconced among astrocytes starting at 28 days. Also by this time large, smoothly contoured, empty secondary cavities appeared, usually rostral and caudal to the epicenter; they did not increase in size or number with time. From 28 days to 17 months postlesion they occurred in 68% of the lesioned spinal cords. The secondary cavity border was composed of cells thought to be astrocytes but, surprisingly, the luminal surface was smooth and lacked basal lamina, in contrast to the primary lesion cavity border. Thus, two types of cavities formed after photochemical lesioning. This lesioning technique may provide an appropriate milieu to better understand aspects of the vexing problem of post-traumatic syringomyelia in the human.

Although cavitary lesions of the spinal cord have been recognized for centuries, only recently have effective, noninvasive imaging techniques allowed antemortem diagnosis of this clinical syndrome. Methods of treatment have not been consistently successful in alleviating or reversing the clinical symptoms caused by these cystic lesions. Incomplete understanding of the underlying pathologic basis for the syringes has impeded the development of effective methods of treatment. This review documents historical considerations regarding clinical observations and experimental studies of this entity and the animal models that have been reported for each of the major types of syringomyelia. Recent studies have suggested that development of a relevant animal model of posttraumatic syringomyelia is imminent. Successful development of an experimental model will not only permit definition of the pathogenesis of cyst formation but also provide methods for testing of therapeutic interventions.

Experimental modeling of spinal cord injury is based mostly on mechanical effects, such as the impact of a weight dropped on the exposed spinal cord. The development of the resultant lesion is influenced by many interactive factors, e.g., the efficiencies of momentum and energy transfer to the cord and their profiles in time, and consequently the histopathologic reproducibility of the lesion is often inconsistent. We describe here a recoilless method that avoids these complications (as well as laminectomy) inherently. The vascular endothelium is injured photochemically, yielding chiefly small-vessel thrombosis and associated vasogenic edema sufficient to generate spinal cord necrosis to predetermined, reproducible degrees. This model is thus intended to simulate the secondary response of the vasculature to mechanical injury In the absence of hemorrhage. The most efficient version of this technique utilizes argon-dye laser excitation of the photosensitizing dye rose bengal at its 562-nm absorption maximum in tissue. With the laser beam focused in the shape of a thin (0.3-mm) line transverse to the spinal column at T8, a narrow zone of necrosis is initially produced. Within 1 week this initial zone expands in volume (length, 6-7 mm) to create a space that, when cleared of cellular debris, is suitable for cell Implantation. In cross section, striking features are the sharp horizontal demarcation between necrosed and viable tissue and the uniform progression of lesion depth as a function of Irradiation time. The necrotic region is bordered dorsally and laterally by a thin rim of viable tissue except at the beam focus; starting at 5 days there is evidence of demyelination in this peripheral region. By 14 days, myelination by oligodendrocytes and Schwann cells begins near this rim; large numbers of Schwann cells enter the dorsal cord at the epicenter, and myelinated axons occupy previously degenerated areas. By 2 months, the initial necrotic area begins to diminish, flattening laterally into clefts. Large, empty cavities develop secondarily with luminal surfaces that are smoothly contoured, as seen by electron microscopy, in contrast to the relatively irregular border of the initial necrotic lesion. Many of these morphological attributes mirror those observed after contusion injury. We anticipate that the uniformity and reproducibility of the photochemical lesion will prove useful in conducting statistically efficient tests of strategies, such as the administration of drugs, trophic factors, or transplanted cells, which are proposed to improve outcome after spinal cord injury.

A clonal, neuronally-differentiating cell line, RN33B, was previously developed by retroviral infection of neural tissue derived from embryonic Sprague-Dawley raphe nuclei with a retrovirus encoding the temperature-sensitive allele of SV40 large T-antigen. In the present study, RN33B cells were transplanted into two target areas of the raphe nuclei, the spinal cord and hippocampal formation, of adult allogeneic hosts. Prior to transplantation, RN33B cells were infected in vitro with a retroviral vector carrying the Escherichia coli lacZ reporter gene and were visualized in vivo using a βgalactosidase immunohistochemical technique. RN33B cells were seen throughout the spinal cord and hippocampal formation of the adult hosts at 15 days post-transplantation. T-antigen-immunoreactive nuclei were detected where RN33B cells were observed, but in much greater numbers than β-galactosidase-immunoreactive cells. Bipolar RN33B cells were found in the spinal cord grey matter. RN33B cells with multipolar morphologies were visualized in the hippocampal and subicular pyramidal cell layers, and also in the dentate gyrus granule cell and polymorph layers, while bipolar RN33B cells were seen in the remainder of the hippocampal formation. The results suggest that immortalized neural cell lines of CNS origin can differentiate in the adult CNS with their ultimate morphology being determined by local tissue signals. We speculate that endogenous neutrophins may significantly influence RN33B cell differentiation in vivo.

A microcomputer-based system employing photoelectric devices to record rat movements and footfalls in the grid walking test paradigm was developed and evaluated. Behavioral data obtained from the system were: distance traversed on the grid, time taken to traverse the distance, number of footfalls, times of footfalls, positions of footfalls, durations of footfalls, and whether the footfall was due to a hind or a fore limb. Validation of the system was performed by comparing the data obtained from the videotape analysis with that obtained from the computerized system. Correlation coefficients between the data obtained from the two methods were found to be 0.92 for one observer, 0.84 for a second observer, and 0.88 with the mean of the two observers. An experimental study in which a group of rats was administered dorsal hemisection lesions of the spinal cord was also conducted. Animals in the lesion group took the same amount of time to cross the runway as the control animals, but made more footfalls per crossing and had longer durations per footfall. The studies validate the capacity of the computerized system to efficiently detect fine locomotory deficits, suggesting that it is a viable tool for the evaluation of neurological dysfunctions in experimental rats.

Two inbred strains have been developed from a cross between SHR and WKY. WK-HTs are hypertensive but not hyperactive, and WK-HAs are hyperactive but normotensive. Together with SHR (that express both traits) and WKY (expressing neither trait) we used four strains to follow correlations of biological changes with the expression of hyperactivity or hypertension. We show that the well known sympathetic hyperreactivity of SHRs to acute stress is associated with the hyperactivity trait and not the hypertension among the four strains. Similarly, the well known ventricular hypertrophy in SHRs is more prominent among the hyperactive strains than the hypertensives. Examination of regional brain amine levels revealed an imbalance in forebrain serotonin transmission in the hyperactive strains, and no significant correlations with hypertension. On the other hand, neuropeptides in brainstem and spinal cord revealed a decrease, in hypertension, in neuropeptide Y and PNMT content of terminals of C1 fibers that innervate the spinal cord sympathetic outflow. Also, the two hypertensive strains showed increased TRH-and proctolin-like immunoreactivity in fibers that innervate the C1 cells in the rostral ventrolateral medulla. These findings illustrate the unique advantage provided by WK-HA and WK-HT strains as additional controls for SHRs in studying hypertension and hyperactivity.

The present study describes the production of a spinal cord lesion which is initiated by vascular occlusion resulting from the interaction between the photosensitizing dye erythrosin B and an argon laser beam. The lesion has characteristics similar to those of the central cavity thought to lead to the production of post-traumatic syringomyelia (PTS) in humans. The present study examines the behavioral and morphological characteristics of this injury over a 28-day period. Histological analysis revealed a cavity extending from the dorsal horns to lamina VIII, with some lateral and ventral pathways being spared. The cavity volume reached a maximum 7 days after lesion induction. Behavioral changes were assessed using six different tests of motor and reflex function (motor function, climbing, waterbath, inclined plane, withdrawal to pain, and withdrawal to extension). Lesioned animals exhibited flaccid paralysis for 3-5 days, which resolved afterward. The photochemically induced cavity should provide a reproducible model for examining the effects of cystic spinal cord injury on locomotor and reflex function.

Bone calcium, histomorphometry, and mechanical strength were evaluated in a model of spinal cord injury. Cortical bone area and rates of formation and apposition at the tibiofibular junction (TFJ) and midshaft of the humerus were measured at 35-42, 42-77, and 77-94 days after transection of the spinal cord. All comparisons were between the animals with a spinal lesion and control animals. A 0.34% difference in the length of the tibia of the two groups of animals was observed, the dry weight of the tibia was 28.4% less in rats with a lesion, and there was no significant difference in the amount of calcium per milligram of bone. At 35 days after surgery, the cortical area in the midshaft of the humerus was slightly less (11%) in rats with a lesion, but by 94 days there was no difference in cortical or medullary area. The final (day 94) cortical area at the TFJ in rats with a lesion was less than that in the controls. Bone formation at the TFJ was similar in both groups in the period of 35-42 days and was similar at 77-94 days in the animals with a lesion, and rates of formation and apposition were greater in the humerus of rats with a lesion; the rates did not differ significantly between groups at either site. At 94 days, trabecular bone area in the tibial metaphysis was 41% less in rats with a lesion. Mechanical parameters were significantly less in the femora, but not the humeri, of rats with a lesion.

The HT4 cell line was derived from infection of a mouse neuroblastoma cell line with a retrovirus that encoded the temperature-sensitive (ts) mutant of SV40 large T antigen. At nonpermissive temperature, HT4 cells differentiated with neuronal morphology, expressed neuronal antigens, synthesized nerve growth factor (NGF) mRNA, and secreted biologically active NGF in vitro. We sought to establish whether transplanted HT4 cells expressed class I major histocompatibility complex (MHC) antigens, a partial requirement for recognition by cytotoxic T lymphocytes (CTL), and thus be susceptible to xenograft rejection. Differentiated HT4 cells expressed marginally detectable levels of class I MHC antigens, but demonstrated higher levels of class I MHC expression after treatment with interferon-gamma. However, HT4 cells were resistant to direct lysis by perforin, the pore-forming protein of CTLs, and thus may have potential use in xenograft experiments. To address whether HT4 cells secrete NGF in vivo, HT4 cells were transplanted into adults rats with unilateral fimbria-fornix transections. A ts cell line derived from P4 cerebellum, BT1, that does not differentiate with neuronal phenotype or synthesize NGF in vitro, was transplanted as a control. Six weeks posttransplant. HT4 cells had integrated into host CNS without forming tumors. In BT1 transplants, the number of medial septal acetylcholinesterase (AChE)-positive cells was reduced to 26-39% of the contralateral control side, depending on the rostrocaudal level. In HT4 transplants, the number of cholinergic septal neurons was 58-78% of the contralateral side. This percentage was significantly (P less than 0.005) greater than that seen with BT1 transplants, indicating that transplanted HT4 cells secrete NGF in vivo and rescue cholinergic septal neurons following fimbria-fornix transection.

A double-peroxidase procedure was used to study the ultrastructural relationships between terminals and fibers containing three putative neurotransmitters and retrogradely identified sympathetic preganglionic neurons (SPNs) located in the intermediolateral cell column (IML) of the rat. SPNs with axons in the cervical sympathetic trunk were retrogradely labeled with horseradish peroxidase. In addition, terminals and fibers containing substance P, enkephalin, and serotonin were detected using immunohistochemistry. Sections containing both retrogradely labeled SPNs and immunoreactive processes were processed for electron microscopy. Ultrastructural examination revealed synaptic contacts between terminals containing each of these three neurotransmitters and retrogradely labeled dendrites from SPNs. Also, immunoreactive terminals were apposed to retrogradely labeled cell bodies. Therefore, these transmitters may alter sympathetic function by their direct action on SPNs.

Glutamate is thought to serve as a major excitatory neurotrans-mitter throughout the central nervous system (CNS)1,2; electrophysiological studies indicate that its action is mediated by multiple receptors. Four receptors have been characterized by their selective sensitivity to N-methyl-D-aspartate (NMDA), kainic acid (KA), quisqualic acid (QA) and 2-amino-4-phosphonobutyric acid (APB)1,3–5. Electrophysiological evidence indicates that these receptors are all present in the rat hippocampus and that the anatomically discrete synaptic fields within the hippocampus exhibit differential sensitivity to the selective excitatory amino acid agents3,6,7. Thus, we have used the hippocampus as a model system to investigate possible subpopulations of 3H-L-glutamate binding sites. By using quantitative autoradiography, the pharmacological specificity of 3H-L-glutamate binding in discrete terminal fields was determined. We report here that there are at least four distinct classes of 3H-L-glutamate binding sites which differ in their anatomical distribution, pharmacological profile and regulation by ions. Two of these sites seem to correspond to the KA and NMDA receptor classes, and a third site may represent the QA receptor. The fourth binding site does not conform to present receptor classifications. None of these binding sites corresponds to the major glutamate binding site observed in biochemical studies8–12.

A rat beta-nerve growth factor (NGF) genomic sequence encoding the entire 3' exon of preproNGF was cloned, and its nucleotide sequence was determined. Rat NGF shows very high homology with other known NGFs in both the prepropeptide and the 3' untranslated regions. The presumptive signal sequence, the cysteine residues important for tertiary structure, possible glycosylation sites, and dibasic amino acids required for proteolytic cleavage to mature NGF are conserved across species. Comparison of the hydrophobicity plots and amino acid sequences revealed an evolutionary divergent domain on the external surface of NGF, which may account for the poor immunologic crossreactivities of the various NGFs. In situ hybridization with a rat-specific oligodeoxynucleotide indicated high levels of NGF mRNA synthesis in both hippocampal granule and pyramidal cell layers. These results are consistent with one role for NGF in the CNS as a neuronally released, retrogradely transported neurotrophic factor for basal forebrain cholinergic neurons.

Serotoninergic (5-HT) neurons derived from the embryonic raphe nuclear area (brainstem, embryonic days (E 16-18) were implanted into the entorhinal cortex of 6-day-old (P6) neonatal rat recipients which had received a fimbria lesion and entorhinal cortex ablation on P3. The hippocampus, dentate gyrus, and the raphe implant area were examined with 5-HT immunohistochemistry 7, 14, 21, 30, and 60 days after implantation. The pattern of 5-HT reinnervation was compared to that of normal and lesioned animals, and to previous studies in which rats received septal or striatal implants. In the hippocampus adjacent to the implant 5-HT-immunoreactive fibers were first observed by 7 days postimplantation and increased in density and in their septotemporal and dorsoventral extent with increasing time postimplantation. Moderately dense fiber networks were diffusely distributed in the hippocampus and dentate gyrus at 30 and 60 days postimplant. Little, if any, indication of lamination was present. Retrogradely labeled neurons (the majority of which contained 5-HT immunoreactivity) were observed in the raphe implant following injections of Fast Blue into the hippocampal formation. A few retrogradely labeled cells did not contain 5-HT, methionine-enkephalin (ME), or substance P (SP) immunoreactivity, although ME- and SP- immunoreactive neurons were observed in the implants. The lamination patterns and the increased density of 5-HT-immunoreactive fibers following a raphe implant into the entorhinal cortex clearly differ from the normal 5-HT pattern and from the patterns of lamination following a striatal or septal implant.

The ultrastructural features of the serotoninergic innervation of the rat dentate gyrus in normal adults and in animals receiving raphe nuclear area transplants was investigated using an antibody to serotonin (5-HT). Neonatal rats received a lesion of the fimbriafornix and entorhinal cortex. Three days later, a portion of embryonic (E-16-18) raphe nuclear area was transplanted to the entorhinal cavity and the animals were allowed to survive for 60 days. Animals were processed for the immunocytochemical localization of 5-HT using the peroxidase—antiperoxidase method. Light microscopic observation showed that 5-HT-containing fibers from transplanted raphe neurons densely innervated the hilar and molecular zones of the dentate gyrus. Electron microscopic analysis showed that 5-HT immunoreactivity was contained only in axons and axon varicosities. There were no differences in the ultrastructural characteristics of axons and axon terminals between normal animals and those which had received raphe transplants. A mixture of both conventional synaptic junctions and non-synaptic axonal swellings were found in both groups.

Publisher Summary This chapter discusses results demonstrating that neurons often contain more than one chemical compound. The different types of coexistence situations are described, including (1) a classical transmitter and one or more peptides, (2) more than one classical transmitter, and (3) a classical transmitter, a peptide, and adenosine triphosphate (ATP). The functional significance of these histochemical findings is at present difficult to evaluate, but in studies on the peripheral nervous system evidence has been obtained that classical transmitter and peptide are coreleased and interact in a cooperative way on effector cells. In addition to enhancement, there is evidence that other types of interactions may occur—for example, the peptide may inhibit the release of the classical transmitter. Also in the central nervous system (CNS), indirect evidence is present for similar mechanisms—that is, to strengthen transmission at synaptic (or non-synaptic) sites and for the peptide inhibition of release of a classical transmitter. Multiple messengers may provide the means for increasing the capacity for information transfer in the nervous system.

A proctolin (PROC)-like peptide was studied immunohistochemically in the hypothalamus, lower brainstem and spinal cord of the rat using an antiserum against PROC conjugated to thyroglobulin. Neuronal cell bodies containing PROC-like immunoreactivity (PROC-LI) were observed in the dorsomedial, paraventricular and supraoptic nuclei of the hypothalamus and in the nucleus raphe magnus, nucleus raphe pallidus, nucleus raphe obscurus and nucleus interfascicularis nervi hypoglossi in the medulla oblongata. Fibers containing PROC-LI were seen in the median eminence and in other hypothalamic nuclei, and in the lower brainstem in cranial motor nuclei including the dorsal motor nucleus of the vagus nerve, the motor trigeminal nucleus, the facial nucleus and nucleus ambiguous, and in lower numbers in the nucleus of the solitary tract and locus coeruleus. Fibers containing PROC-LI were also located in the spinal cord, in the intermediolateral cell column at thoracic levels and in the ventral horns at all levels of the spinal cord. After transection of the spinal cord, all PROC-immunoreactive fibers below the lesion disappeared. Following injection of Fast blue into the thoracic spinal cord, retrogradely labeled cells in the nuclei raphe pallidus, obscurus and magnus and nucleus interfasciculari nervi hypoglossi were seen to contain PROC-LI. PROC-LI had a similar distribution as thyrotropin-releasing hormone (TRH)-LI in the above-mentioned areas and coexistence of TRH-LI and PROC-LI was shown in cell bodies in the hypothalamus and medulla oblongata. PROC-LI could also be shown to coexist with 5-hydroxytryptamine (5-HT)-LI in neuronal cell bodies in the lower brainstem. The results demonstrate the occurrence of a PROC-like peptide in the mammalian nervous system, and these neurons seem to be at least largely identical to previously described TRH systems. A possible involvement of the PROC-like peptide in spinal motor control is discussed in relation to the well-established role of PROC in control of motor behavior in insects and invertebrates.

High levels of NGF have recently been detected in cerebral cortex and hippocampus, and it was suggested that NGF supports cholinergic, basal forebrain neurons. The present study directly examined whether NGF levels are altered in the neonatal hippocampus following cholinergic denervation by transection of the fimbria. Ten days after transection, hippocampal cholinergic innervation, as assessed by AChE histochemistry and CAT immunohistochemistry, was decreased, and both hippocampal NGF mRNA and protein were elevated about 50%. This indicates possible lesion-induced transcriptional control of neonatal hippocampal NGF levels. This increase was specific to lesions of cholinergic systems, as entorhinal cortex ablation, which removes other afferent fibers to the hippocampus, did not cause a similar increase. At 30 d after fimbria transection, hippocampal NGF mRNA and protein did not differ from control levels, but the decrease in AChE and CAT staining persisted. Peripheral sympathectomy carried out in the adult rat resulted in 2- to 5-fold increases in NGF protein levels in heart atrium and ventricle, as well as submandibular gland, with no concomitant increase in NGF mRNA. Therefore, the control of NGF levels in the adult PNS is probably posttranscriptional. Our results strongly suggest that NGF is involved in the regulation of central cholinergic neurons and is transiently elevated in the neonatal hippocampus following cholinergic lesion.