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Von Ebner's glands of the rat are minor salivary serous glands in the posterior portion of the tongue. They secrete two digestive enzymes, lingual lipase and amylase. In this investigation, circadian rhythm in feeding was established under a normal 12 h light/12 h dark cycle, with the rats eating primarily during the dark period. At lights on, the size of the acinar cells and the area of the inclusive secretory granules, and the amount of digestive enzyme activity (lingual lipase and amylase) remaining in the gland was significantly less than in the mid-afternoon, after very little daylight food consumption. However, after 7 days of continuous light the circadian rhythm was altered: the food consumption during the normal night-time hours (5 p.m. to 8 a.m.) went from 88% of total 24 h food consumption to 45%, and during normal daylight hours (8 a.m. to 5 p.m.) from 12% to 55%. These changes were correlated with histometric findings of a near reversal of the areas of acinar cells and secretory granules of a.m. and p.m. samples under continuous light. Lingual lipase activity in the glands went from 35% under 12 h light to 61% under continuous light in the a.m. and from 65% to 39% in the p.m. Amylase activity also showed nearly a reversal in activity remaining in the gland, from 36% at 12 h light to 58% at 24 h light in the a.m. and 64% to 41% for the p.m. samples. These results indicate that the von Ebner's glands of the rat have a circadian rhythm of secretion and storage of secretory proteins that is subject to light entrainment similar to that seen in other exocrine glands such as the parotid and pancreas.

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BACKGROUND: Invasion and metastasis is aided by the secretion of guanidinobenzoatase, that cleaves the link peptide to fibronectin, and urokinase plasminogen activator (uPA), which initiates a molecular cascade to activate plasmin and collagenases. This process permits malignant cell migration through the extracellular matrix. MATERIALS: Original human astrocytomas were examined for guanidinobenzoatase and uPA. Suspensions of high-grade human astrocytomas were xenografted into pockets in host cerebral cortex for 1-7 days. RESULTS: A class of guanidinobenzoatase positive cells was observed in the original human astrocytomas and in tumor masses formed in the implantation pocket and around blood vessels. Secondary foci containing guanidinobenzoatase positive cells formed around blood vessels and individual positive astrocytoma cells migrated on the glia limitans along parallel and intersecting nerve fiber fascicles and the corpus callosum. uPA and GFAP were colocalized with guanidinobenzoatase. CONCLUSION: The high-grade astrocytomas reestablish themselves and maintain their characteristics as a tissue although grafted as individual cells.

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The following series of experiments explores the post-xenografting differentiation of a naturally occurring, non-neuronal cell cultured from the leptomeninges of an 84-year-old woman. In culture, flat process-bearing human cells from the leptomeninges were positive for GFAP and 200 kDa neurofilament protein (negative for 68, 160 kDa neurofilament protein). The C3 spinal cord was exposed in 30 adult athymic rats. The hindlimb dorsal columns were transected at C3 and the nerve fibers aspirated to form a pocket, into which 10(6) fast blue-labeled, human leptomeningeal-derived cells were placed. The C3 spinal cord was studied immunohistochemically over 60 days. Three days later the dorsal horn contained fast blue-GFAP-positive astrocyte-like cells that were negative for neurofilament protein. By 7 days, large, process-bearing, fast blue-GFAP-positive (neurofilament protein-negative), astrocyte-like cells joined the native astrocytes of the pia-glia membrane and were in the gray matter of the spinal cord. Some of these astrocyte-like cells were also positive for the human specific histocompatibility complex, HLADR. These data extend the age, species and tissue of origin for pluripotential cells for CNS transplantation.

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BACKGROUND: Bile acid exposure produces cellular hypercalcemia in gastric and hepatic cells. It is not known, however, whether this event contributes to cell injury or if it results from passive equilibration of calcium ion concentrations across the membranes of irreversibly damaged cells. This study was performed to determine whether the cellular hypercalcemia produced by bile acid exposure in gastric cells is reversible and to determine whether the source of this hypercalcemia is from intracellular stores of calcium, extracellular sources, or both. METHODS: Cytosolic free calcium concentrations ([Ca]i) were measured in rabbit gastric mucosal cells that had been loaded with the intracellular probe FURA-2. Measurements were performed in suspensions of dispersed cells by using standard spectrofluorometry and in primarily cultured cells by using fluorescence videomicroscopy. Measurements were made before and after exposure to 0.2, 0.5, and 1.0 mmol/L deoxycholic acid (DC). These measurements were made in the presence of 1 mmol/L extracellular calcium and in the absence of any extracellular calcium (0.5 mmol/L EGTA). RESULTS: In experiments with dispersed cells and spectrofluorometry, [Ca]i increased from a pretreatment level of 194 +/- 8 nmol/L to 396 +/- 21 nmol/L within 3 minutes of exposure to 0.2 mmol/L DC. When these cells were washed and resuspended in DC-free medium, [Ca]i] decreased to 180 +/- 5 nmol/L. In experiments with cultured cells and fluorescence videomicroscopy, rapid, reversible hypercalcemia was observed after exposure to 0.5 and 1.0 mmol/L DC. Removal of extracellular calcium from the incubating medium reduced both the magnitude and duration of the observed hypercalcemia. CONCLUSIONS: These data show that the cellular hypercalcemia that accompanies DC-induced injury in gastric cells is a reversible event. The initial increase in [Ca]i appears to come from both intracellular and extracellular sources, although sustained hypercalcemia requires a source of extracellular calcium. As a reversible event, cellular hypercalcemia may be an important pathophysiologic feature of bile acid induced injury of the upper gastrointestinal tract.

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Individual astrocytoma cells expressing a cytoplasmic form of p185c-neu migrated along basement membrane lined surfaces after xenografing fresh low or high grade human malignant astrocytomas into host rat brain. We now study the migratory capacity of fresh human malignant astrocytoma cells seeded on hydrated gel wafers composed of artificial basement membrane or collagen I, a normal and lesion-related CNS extracellular matrix component. Approximately 10(7) mechanically disrupted cells (with small clumps) of 3 fresh low grade and 6 fresh high grade astrocytomas were seeded on the surface of artificial basement membrane and collagen I wafers (11 x 16 mm). The wafers were then prepared for scanning electron microscopy and immunohistochemistry at 1, 3, 5, and 7 days after seeding. Regardless of tumor grade, a morphologically similar class of cells was observed to migrate through collagen I gels in 24 hours and 0.5-1.5 mm into artificial basement membrane gels in 7 days. Immunohistochemistry revealed that the migrated cells from low and high grade astrocytomas were positive for glial fibrillary acidic protein (GFAP)) and expressed cytoplasmic human-specific p185c-neu. These data indicate that fresh human malignant astrocytoma cells that contain GFAP and express cytoplasmic p185c-neu have a high degree of migratory capacity and could be the cell in the tumor involved in intraparenchymal metastasis and poor patient survival in high grade astrocytomas of the human brain.

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Fresh suspensions of human glioblastoma multiforme were preincubated in the plant lectin Phaseolus vulgaris leucoagglutinin (PHAL) and implanted into cortical pockets in adult rat brain. Brains were investigated periodically over 30 postoperative days and the migration of the human glioblastoma cells was traced with anti-PHAL immunofluorescence or the overexpression of human specific p185c-neu a specific marker of a class of human malignant astrocytoma cells. The principal pathway of migration of the implanted human cells in the rat brain was ventrally through cortical gray matter and into the corpus callosum, with rapid lateral distribution in this and other parallel and intersecting white matter fascicles. Human glioblastoma cells also migrated on basement membrane lined blood vessels, pia-glia membrane and spaces of Virchow-Robin, as well as the subependymal space of the ventricles. These paths of migration of human glioblastoma cells in the rat brain are consistent with the pathways of spread of glioblastoma in the human brain as described by Scherer over 50 years ago, indicating that multifocal malignant astrocytomas have common migratory pathways in mature mammalian brain.

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The immunoglobulin (IgG, IgM, IgA) content of normal, reactive and migrated rat astrocytes was studied in lesioned adult rat cortex and after fetal cortex grafts. Implantation pockets were aspirated in the somatomotor cortex (under bregma) of adult Sprague-Dawley rats. A fetal (E14) rat hemicortex incubated in the plant lectin Phaseolus vulgaris leucoagglutinin (graft premarker) was placed in the aspiration pocket or the pocket left empty (control). Sections of brain were immunohistochemically double labeled for immunoglobulins-GFAP or Phaseolus vulgaris leucoagglutinin-GFAP 3-60 days postoperative. Mature or fetal astrocytes in situ did not contain immunoglobulins. In pocket-only animals, individual reactive astrocytes lining and subjacent to the pocket were positive for rat IgG, IgM and IgA (3-60 days). In grafted animals, graft derived astrocytes (Phaseolus vulgaris leucoagglutinin-GFAP positive) were intermingled with host reactive astrocytes (Phaseolus vulgaris leucoagglutinin negative) lining and subjacent to the pocket. Both classes of astrocytes contained immunoglobulins IgG, IgM and IgA. Immunoglobulin positive graft derived astrocytes migrated into the corpus callosum, cingulum and habenula. These results demonstrate that astrocytes sequester immunoglobulins and probably other serum proteins as a critical function in restoring homeostasis to the injured or diseased nervous system.

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Nerve growth factor (NGF) immunoreactivity in the nucleus gracilis of the medulla was quantitated for 90 days after aspiration of the C3 spinal hindlimb dorsal columns of 36 adult rats. Half the lesioned animals were a lesion-only group. The remaining lesioned animals received an immediate graft of two 1.0-mm pieces of 14 day gestation fetal rat cervical spinal cord (prelabeled with Phaseolus vulgaris leucoagglutinin) into the aspiration pocket (graft group). There were 3 normal controls. Groups of animals were analyzed at 7, 14, 21, 30, 60, and 90 days. At 90 days, NGF immunoreactivity was significantly elevated in the nucleus gracilis of lesion-only animals. This increase in NGF immunoreactivity was augmented in glial end-feet surrounding neurons and was also observed in the cytoplasm of astrocytes and some neurons. Previous experiments have shown that the cluster neurons of the nucleus gracilis undergo atrophy at this time with a concomitant decrease in hindlimb placement. NGF immunoreactivity (90 days) in grafted animals, however, was significantly less than in lesion-only animals (P < 0.05) but remained significantly elevated above control animals (P < 0.05). Unlike in lesion-only animals, there were no NGF positive neurons in the nucleus gracilis of grafted animals. Previous experiments have shown that astrocytes from fetal spinal cord grafts migrate to the nucleus gracilis, maintain cluster neuron cell size, and improve hindlimb placement at 90 days. The present data indicate that modulation of detrimental increases in NGF appeared to be a mechanism by which migrated fetal astrocytes can be used as a system for cell therapy.

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Fetal basal ganglia astrocytes and C6 glioma cells were plated on the surface of 1.5 cm thick hydrated collagen I wafers. Both cell types migrated through the entire thickness of the wafer within 1 day after plating. The collagen in the wafer was digested and the fine collagen I fibrils were clumped into large strands. By 2-3 days, the collagen strands were digested from the wafers and replaced by a mass of fetal astrocytes or C6 cells joined by their processes. The collagen I digestion and cell migration suggested protease production. In a second series of experiments, cultured C6 cells and E14 fetal astrocytes were immunohistochemically stained for the presence of plasminogen activators as an index of protease production. Both tissue (tPA) and urokinase (uPA) types were observed. Fetal astrocytes and C6 cells were also positive for guanidinobenzoatase, a serine protease associated with migrating cells. These data demonstrate that rapid migration of the cells on and through collagen I fibrils is concomitant with expression of plasminogen activators and proteases which can either activate or function as collagenases and release the cells from the substrate.

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Scanning electron microscopic studies revealed that the cell bodies of cultured C6 cells were densely covered with villi and that membrane blebs were rare. Exposure to a hydroxyl radical generating system resulted in rapid ultrastructural changes. Within 2.5 minutes, 98% of the cells lost their villi. The number of blebbed cells increased during the first 7.5 minutes of exposure, until virtually all cells were blebbed. After 20 and 30 minutes of exposure, the plasma membrane of the blebs ruptured, resulting in the escape of cell contents and cell necrosis. Trypan blue exclusion, a measure of cell viability, decreased between 1 and 7.5 minutes of exposure. No further significant decline in viability was observed for the remainder of the experiment. The hydroxyl radical generating system also initiated a rapid onset of membrane lipid peroxidation, as measured by accumulation of thiobarbiturate reactive material. After a short lag period, thiobarbiturate reactive material increased rapidly, with maximum lipid peroxidation occurring by 20 minutes. Increasing the concentration of the radical generating system components shortened the time course but not the level of maximal thiobarbiturate reactive material production. These data suggest that plasmalemmal lipid peroxidation plays a role in rapid morphological changes and necrosis that occur after free radical insult.

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Cortically homografted C6 glioma-astrocytoma cells both invade the rat host brain as a mass and migrate as individual cells. In contrast, fetal astrocytes derived from homografted whole pieces of fetal cortex migrate only as individual cells throughout the brain of the rat but are not capable of invasion. Our experiment explored the migratory capacity (over 7 days) of cultured purified fetal astrocytes and C6 cells after seeding 10(6) cells on a hydrated artificial basement membrane wafer (Matrigel). The artificial basement membrane wafer was not a suitable substrate for the growth of cultured fetal astrocytes. In contrast, C6 cells migrated as individual cells from the surface of the wafer into the substrate. Individual C6 cells migrated 1.8 mm in the first 4 days and then ceased migration. The C6 cells were observed at the base of a digestion tube that extended from and was open to the surface of the wafer. At 3 days, micropockets were observed to form around each C6 cell at the base of each tube. By 7 days, the majority of pockets observed were large and contained several C6 cells. These multiple cell groups appeared to be progenitors of tumor masses. These data indicate that C6 glioma-astrocytoma cells, which in vivo appear to be a model for glioblastoma multiforme, primarily migrate as individual cells through artificial basement membrane and secondarily form tumor masses. Progenitor tumor masses form by coalescence of individual C6 cell micropockets or the division of a single cell in an individual micropocket.

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Cultured C6 glioma cells were prelabeled with the plant lectin Phaseolus vulgaris leuco-agglutinin (PHAL) and grafted as a cell suspension (10(6) cells in 5.0 microliters) into freshly made cortical implantation pockets in adult host rats. Animals were killed 1-21 days post-implantation (DPI). The brains were removed, dehydrated, embedded in paraffin and sectioned at 8 microns. Paraffin sections were processed for light level immunofluorescent double labeling for PHAL, a marker for graft derived cells, and glial fibrillary acidic protein (GFAP), a specific marker for C6 glioma cells and astrocytes. Cells positive for both PHAL and GFAP were graft-derived C6 cells. By 7 DPI a large mass developed which extended above the surface of the brain and invaded (displacement of host tissue by a cell mass) the host parenchyma. This mass increased in size over the next 14 days. The invading tumor mass contained double labeled cells at all time periods examined. In addition to the invasion process, grafted C6 cells spread through the host parenchyma by migration (movement of single cells). Individual graft-derived C6 (GFAP/PHAL positive) cells migrated into host cortex surrounding the implantation pocket, corpus callosum ventral to the implantation pocket, ipsilateral internal capsule and bilaterally in the habenula.

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In a 'double blind' study, 2 series of adult rats were trained to traverse a narrow bar or a horizontal ladder for a water reward. Hindlimb placement (measured as hindlimb foot slips) of the subjects trained on the narrow platform was ranked. The subjects traversing the ladder were videotaped and the number of hindlimb slips counted. After reaching criterion (10 complete traverses on each of 2 consecutive days), all animals had the fasciculus gracilis (FG) of the third cervical spinal cord segment (C3) aspirated to sever hindlimb dorsal column afferents. After aspiration of C3FG the subjects were randomly placed in an aspiration-only (controls) or aspiration + graft group. All grafts were prelabeled with the plant lectin Phaseolus vulgaris leucoagglutinin (PHAL) as a graft-derived cell marker. The grafts were either unoriented whole pieces of E14 fetal spinal cord or 105 purified, cultured, E14 fetal spinal cord astrocytes. Subjects were tested at intervals over 90 days. Aspiration of C3FG and a graft of whole pieces of E14 fetal spinal cord resulted in a statistically significant improvement in hindlimb placement at 21 and 90 days (P < 0.05) when compared to controls. In contrast, animals with cultured, purified, E14 fetal spinal cord astrocyte grafts had significantly worse (P < 0.05) hindlimb placement than controls. Immunohistochemical double staining for PHAL and glial fibrillary acidic protein in the same cell showed that astrocytes from both types of grafts migrated to the nucleus gracilis (NG) of the medulla of the host. The grafted fetal astrocytes from the whole piece grafts prevented denervation atrophy of the host NG neurons, whereas cultured grafted fetal astrocytes did not demonstrate this effect. After grafting donor tissue that contained astrocytes into the injured spinal cord there were two accompanying classes of astrocytes. One class derived from whole piece, E14, fetal spinal cord grafts migrated to the NG of the host, prevented atrophy of host NG cluster and interneurons, and improved hindlimb placement. The other class, derived from cultures of E14 fetal spinal cord astrocytes, migrated to the NG of the host, failed to maintain the size of cluster neurons and interneurons of the host NG, and resulted in a greater hindlimb placement deficit when compared to controls. These data suggest that improvement of hindlimb placement due to graft-derived fetal astrocytes in injured host spinal cord was due to the ability of the astrocytes to maintain host neurons and neuronal networks.

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C6 tumor cells (10(6] were grafted as suspensions into freshly made implantation pockets in rat host cerebral cortex. Specimens were prepared for transmission and scanning electron microscopy 1 to 7 days postimplantation (DPI). By 3 DPI vacuolated C6 cells had migrated on or invaded the host brain. C6 cells were observed on the glia limitans on the surface of the brain, in the corpus callosum, subependymal space, and perivascular space and had invaded the cortex under the implantation pocket. In addition to the tumor mass that was observed under the implantation pocket, by 7 DPI individual C6 cells had migrated into the corpus callosum and internal capsule. Migrated C6 cells were observed in a perineuronal position in the hippocampus and other gray matter structures inferior to the corpus callosum. Micropockets were found around each C6 cell and the processes of these cells had replaced host parenchyma. The preferred routes of migration were on basal lamina and parallel and intersecting nerve fiber bundles. Invasion occurred through gray and white matter. The movement of homografted C6 cells in the brain suggests that these cells actively migrate as individual cells in addition to invading as a mass.

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The cerebral cortices from 14-day gestation rat embryos were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and homografted as a cell suspension into host thoracic spinal cord. Animals were sacrificed at 7, 14, 21 and 30 days postimplantation (DPI). Paraffin sections of cervical, thoracic and lumbar spinal cord were double-labeled for the presence of glial fibrillary acidic protein (GFAP) a specific marker for astrocytes, and PHAL, utilized as a marker for graft-derived cells. PHAL-GFAP positive cells were found throughout the thoracic spinal cord at all time periods, indicating that grafted astrocytes migrated along all 3 axes of the host spinal cord (rostral-caudal, dorsal-ventral and left-right). At 30 DPI, graft-derived astrocytes were found in host cervical and lumbar spinal cord. There appeared to be a minimal delay in the onset of migration.

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The fasciculus gracilis (FG) of the third cervical spinal cord segment (C3) of adult rats was aspirated and fresh whole pieces of rat 14-day gestation cervical spinal cord, prelabeled with Phaseolus vulgaris leucoagglutinin, immediately placed in the pocket of one-half the operates. One to three months later there were no transplant derived rostro-caudal nerve fibers in the C1 or C2 segment of the aspiration-only or aspiration-graft FG. However, the neurons of the nucleus gracilis of the host medulla of the aspiration-only group were significantly atrophied whereas the neurons of the aspiration-graft group were not statistically different from normal. There were normal numbers of neurons in all groups. Using immunohistochemical double labeling for prelabeled astrocytes with PHAL and GFAP, graft-derived astrocytes were found in the nucleus gracilis of the host medulla. These data indicate that the trophic action of spinal graft-derived astrocytes maintained neurons in the medulla of the host nucleus gracilis.

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Fresh xenografted human malignant astrocytoma cells migrate throughout the host rat brain. Cells from three Grade 3 human malignant astrocytomas were prelabeled with Phaseolus vulgaris leucoagglutinin (PHAL) and then xenografted into implantation pockets in rat host cerebral cortex. The human malignant astrocytoma cells in the host brain were immunocytochemically double-labeled for the presence of PHAL, which is used as a marker for graft derived cells, and either glial fibrillary acidic protein (GFAP), a specific marker for astrocytes and astrocytoma cells, or apolipoprotein E (APOE) 7 days, 14 days, 21 days, and 1 month later. Fresh human malignant astrocytoma cells (Grade 3 and 4) contained APOE and GFAP. The xenografted cells preserved APOE and GFAP in the host. PHAL double-labeled human malignant astrocytoma cells were found on the glia limitans along the entire circumference of the brain, in the corpus callosum, internal capsule, entopeduncular nucleus, optic tract, and median eminence. In addition, astrocytoma cells were observed in the cingulum, habenula, arcuate, and supraoptic nucleus. Astrocytoma cells entered the space of Virchow-Robin, migrated along parenchymal blood vessels and between the ependymal and subependymal layers of the third and lateral ventricles. APOE was a consistent marker for the migrating human malignant astrocytoma cells, but not an exclusive marker of the xenografted cells, since host rat reactive astrocytes also expressed APOE.

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Fresh cells from two grade 3 human malignant astrocytomas were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and then xenografted into freshly made implantation pockets in rat host cerebral cortex. Animals were sacrificed at 7, 14, 21 days, and 1 month postimplantation (DPI). Paraffin sections were double-labeled for the presence of glial fibrillary acidic protein (GFAP), a specific marker for astrocytes and differentiated astrocytoma cells, and PHAL, utilized as a marker for graft-derived cells. Grafted human astrocytoma cells were found on the glia limitans along the entire circumference of the brain, in the corpus callosum, internal capsule, entopeduncular nucleus, optic tract, and median eminence. In addition, astrocytoma cells were observed in the cingulum, habenula, arcuate, and supraoptic nucleus. Astrocytoma cells entered the spaces of Virchow-Robin, and migrated along parenchymal blood vessels and between the ependymal and subependymal layers of the third and lateral ventricles. The corpus callosum was a major migration route for the astrocytoma cells. The presence of basal lamina or parallel nerve fiber bundles was a common factor for these migration routes. The migration of the human astrocytoma xenografted cells in the rat brain followed the spread of human malignant astrocytomas in the human brain and is a valuable basic science tool in brain cancer research.

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Neuropeptide receptors were visualized in homografts of fetal cortex (E14) into adult rat cortex (immediate or 7 day delay) and spinal cord using in vitro autoradiographic techniques to explore the expression of peptide receptors in the same graft tissue in different central nervous system implantation sites. Receptors for bombesin (BN)-like peptides developed in the grafts by 3 weeks postimplantation regardless of location or age of implantation pocket in host. After 4 weeks, the density of BN receptors was confined to the graft. In grafts to spinal cord, however, high densities of BN-like receptors were not confined to the graft but were distributed throughout the spinal cord. In contrast, the density of vasoactive intestinal polypeptide (VIP) and substance P (SP) receptors was moderate and low to undetectable in the fetal grafts. The development of the peptide receptors studied was graft donor tissue specific since they were not altered by central nervous system implantation site.

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