Selective hippocampal lesions differentially affect the phenotypic fate of transplanted neuronal precursor cells.

RN33B cells, a CNS-derived neuronal precursor cell line, transplanted into normal adult rat hippocampus can survive and morphologically differentiate with their ultimate morphology dependent on the integration site. This study examined the differentiation and structural integration of RN33B cells transplanted into the lesioned adult hippocampus. Pyramidal neurons of the CA1-3 regions or granular neurons in the dentate gyrus were preferentially destroyed by unilateral intraventricular kainic-acid or intradentate colchicine injections, respectively. One week after the lesion, a suspension of undifferentiated beta-galactosidase (beta-gal)-labeled RN33B cells was stereotaxically transplanted into the lesioned or the contralateral hippocampus. After 5-7 weeks, sections of the recipient brains were analyzed by toluidine blue staining and immunohistochemistry for beta-gal, GFAP, and OX-42. A reactive gliosis was observed on the lesioned side which persisted up to 7 weeks postlesion (the latest time point examined). RN33b cells survived in the lesioned hippocampus and assumed variable morphologies depending on the hippocampal layer into which they integrated. Only RN33B cells located in intact or partially damaged cell layers or in the unlesioned contralateral hippocampus differentiated with morphologies similar to those of endogenous neurons characteristic of those layers. Cells located in layers completely depleted of endogenous neurons assumed bipolar morphologies or sent out multiple processes with no structural polarity, unlike the neuronal morphologies characteristically seen in intact hippocampal cell layers. These data suggest that the presence of some endogenous neurons and partially conserved cytoarchitectural organization are essential for immortalized neuroepithelial precursor cells to differentiate into region-specific neuronal cell types.

The adult CNS retains the potential to direct region-specific differentiation of a transplanted neuronal precursor cell line.

The chronic survival and differentiation of the conditionally immortalized neuronal cell line, RN33B, was examined following transplantation into the adult and neonatal rat hippocampus and cerebral cortex. In clonal culture, differentiated RN33B cells express p75NTR and trkB mRNA and protein, and respond to brain-derived neurotrophic factor treatment by inducing c-fos mRNA. Transplanted cells, identified using immunohistochemistry to detect beta-galactosidase expression, were seen in most animals up to 24 weeks posttransplantation (the latest time point examined). Stably integrated cells with various morphologies consistent with their transplantation site were observed. In the cerebral cortex, many RN33B cells differentiated with morphologies similar to pyramidal neurons and stellate cells. In the hippocampal formation, many RN33B cells assumed morphologies similar to pyramidal neurons characteristic of CA1 and CA3 regions, granular cell layer neurons of the dentate gyrus, and polymorphic neurons of the hilar region. Identical morphologies were observed in both adult and neonatal hosts, although a greater percentage of beta-galactosidase immunoreactive cells had differentiated in the neonatal brains. These results suggest that RN33B cells have the developmental plasticity to respond to local microenvironmental signals and that the adult brain retains the capacity to direct the differentiation of neuronal precursor cells in a direction that is consistent with that of endogenous neurons.

Syringomyelia: clinical observations and experimental studies.

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.

Characterization of photochemically induced spinal cord injury in the rat by light and electron microscopy.

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.

Changes in bone in a model of spinal cord injury.

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.

Effect of grafting order on innervation of spinal cord transplants by grafted locus coeruleus neurons in oculo.

Fetal brain stem containing locus coeruleus/subcoeruleus (hereafter referred to as LC) and thoracic spinal cord (SC) were sequentially allografted into the anterior eye chamber of adult Sprague-Dawley albino rats creating two groups: (1) LC graft followed after 11 weeks by an SC graft (LC-SC); (2) SC graft followed after 11 weeks by an LC graft (SC-LC). The cografts were allowed to mature in oculo for 15-18 months. After sacrifice, the grafts were processed for the immunohistochemical localization of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (D beta H) and the extent of fiber ingrowth into the SC graft was measured using computerized image analysis. TH- and D beta H-immunoreactive fibers were found to innervate the SC cograft in five of the six SC-LC graft combinations. The innervation was abundant, but uneven in distribution. The average density of TH-immunoreactive fibers (derived from the LC graft) was 8.29% of the total cross-sectional area of the SC graft neuropil. In contrast, the innervation of the SC graft neuropil was very sparse in the LC-SC graft combinations, with an uneven distribution and an average density of TH-immunoreactive fibers in the SC graft of only 2.28% of the cross-sectional area of the SC graft neuropil. The results support earlier studies of intraspinal grafting of LC neurons, in that embryonic LC neurons upon grafting and during ontogenetic fiber growth are capable of innervating mature spinal cord neuropil devoid of its normal catecholaminergic innervation. However, embryonic spinal cord tissue is a poor stimulant to reinitiate terminal fiber growth from mature LC neurons, in contrast to several other LC target areas such as the cerebral cortex and hippocampus.

Multiple putative neuromessenger inputs to the phrenic nucleus in rat.

Immunohistochemical reactions for 12 putative neuromessengers combined with retrograde labeling of phrenic motoneurons identified seven neuromessengers (5-hydroxytryptamine, substance P, thyrotropin-releasing hormone, methionine enkephalin, cholecystokinin, galanin, neuropeptide Y) located within terminal varicosities in the phrenic nucleus. The degree of terminal labeling in the phrenic nucleus varied depending on the peptide. Substance P, thyrotropin-releasing hormone and methionine enkephalin were each tested for colocalization with 5-hydroxytryptamine within terminal varicosities in the phrenic nucleus, and the coincidence of double-labeling varied for each peptide. These results indicate that phrenic motoneurons are subject to modulation by many peptide neuromessengers that may alter their responsiveness to primary excitatory and inhibitory inputs.

Transplantation of a temperature-sensitive, nerve growth factor-secreting, neuroblastoma cell line into adult rats with fimbria-fornix lesions rescues cholinergic septal neurons.

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.

Photochemically induced cystic lesion in the rat spinal cord. I. Behavioral and morphological analysis.

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.

Rat beta-nerve growth factor sequence and site of synthesis in the adult hippocampus.

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.

Locus coeruleus neurons in the rat containing neuropeptide Y, tyrosine hydroxylase or galanin and their efferent projections to the spinal cord, cerebral cortex and hypothalamus.

The efferent projections of locus coeruleus neurons which contain neuropeptide Y-, tyrosine hydroxylase- or galanin-like immunoreactivity were investigated using the indirect immunofluorescence technique combined with the retrograde transport of the fluorescent substance Fast Blue. Four groups of rats received injections of Fast Blue: (1) bilaterally into the mid-thoracic spinal cord (T6-T7); (2) unilaterally into the low cervical spinal cord (C4-C5); (3) unilaterally into the paraventricular, periventricular and dorsomedial hypothalamic nuclei; and (4) unilaterally into five sites in the cerebral cortex (frontal, cingulate and striate cortex). Efferent projections to the spinal cord, hypothalamus and cerebral cortex from neuropeptide Y-, tyrosine hydroxylase- and galanin-containing locus coeruleus cells were observed. A higher percentage of the peptidergic locus coeruleus neurons projected to the hypothalamus than to the spinal cord or cerebral cortex. The distribution and morphology of the neuropeptide Y- and galanin-containing neurons in the locus coeruleus were also investigated. Neuropeptide Y-like immunoreactivity and galanin-like immunoreactivity were found in small, medium and large multipolar neurons, as well as in fusiform locus coeruleus cells. The neuropeptide Y- and galanin-immunoreactive neurons were found throughout the locus coeruleus. In the caudal locus coeruleus, they were primarily located in the dorsal portion. Neuropeptide Y-like immunoreactivity and galanin-like immunoreactivity were only seen in a few tyrosine hydroxylase-positive neurons of the subcoeruleus group. The data show that the peptide-containing locus coeruleus neurons have efferent projections to the spinal cord, hypothalamus and cerebral cortex. The locus coeruleus may be divided into functional subdivisions dependent on the region of the locus coeruleus, the neurotransmitter/neuropeptide(s) contained within the neurons and their efferent projections.

Postnatal development of the serotonin innervation of the hippocampus and dentate gyrus following raphe implants.

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.