Differential distribution of immunoreactive angiotensinogen in the hind-brain and spinal cord of neonatal and adult rats

The present communication deals with the cytochemical localization of angiotensinogen (ATG) immunoactivity in the hind-brain and spinal cord of neonatal (1-day-old) and adult (3-month-old pregnant) female rats. In the neonatal hind-brain, the immunoreactive cells were more numerous than in that of adult rats. In the adult rat hind-brain, the number of ATG-positive cells was quite limited in each nucleus. Further, in some nuclei, only neurons or neuroglia were positive, while in others the immunoactivity was observed in both the components. Spinal cords of neonatal rats showed a few undifferentiated ATG-positive cells in the grey matter. Contrary to this, the spinal cord of adult animals contained numerous immunoreactive glial cells in the grey matter, fasciculus cuneatus and fasciculus gracilis. Immunoactivity in the neurons was localized in the Nissl bodies.     

Recent advances in neural cell culture

Cells isolated from the avian and mammalian central and peripheral nervous system and cultured in vitro provide an opportunity to study in situ properties of neurons and glial cells under relatively simple and carefully controlled conditions. Since Harrison's success in maintaining in vitro embryonic frog spinal cord 80 years ago, neural tissue culture has developed into an important and versatile discipline of neuroscience. The techniques developed in the past fall into four broad classes: Explant cultures, which are explanted from specific neuroanatomic loci to substrates as small tissue fragments. Dissociated cell cultures, which involve the seeding of enzymatically or mechanically dispersed cells on various attachment substrates. Reaggregate cultures, which require re-association of dissociated cells into small aggregates. Purified cell populations, which are prepared by the isolation of different cell types by gradient centrifugation or other separation techniques. These cultures have been utilized in studying various aspects of brain development and function. In this review several areas of significant and stimulating development in neural cell culture have been documented. They include formulation of serum-free medium, effects of growth factors, utilization of cell type-specific markers, and isolation and culture of purified neuronal/glial cells.     

Simultaneous immunocytochemical visualization of bromodeoxyuridine and neural tissue antigens.

5'-Bromodeoxyuridine (BrdU) is a thymidine analogue which can be detected by monoclonal antibodies (MAb). We have developed a method for the simultaneous visualization of BrdU and a wide range of neural antigens in paraformaldehyde-fixed brain sections. Pregnant mice were injected intraperitoneally with a single pulse of BrdU. Young adult offspring were processed for immunocytochemistry following a double immunoperoxidase sequence. BrdU was detected using diaminobenzidine (DAB) intensified with nickel ammonium sulfate and neural antigen-containing elements were visualized with DAB alone. BrdU-positive nuclei and tissue antigen-immunoreactive cells were easily differentiated. Furthermore, double-labeled cells characterized by the presence of a black immunoreactive nucleus surrounded by a brown immunopositive cytoplasm were unambiguously recognized. Satisfactory results were obtained using either MAb or polyclonal antibodies against a variety of cell antigens, including neuropeptides, CA++ binding proteins, and cytoskeletal components of the glial cells. The method reported here permits analysis of the neurogenesis and proliferation of subsets of neurons and glial cells, identified by immunocytochemical markers.     

Dissociated Neurons and Glial Cells Derived from Rat Inferior Colliculi after Digestion with Papain

The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode–tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue–material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3_5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2×10⁴ cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.     

Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins

In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality.     

Mixed primary culture and clonal analysis provide evidence that NG2 proteoglycan-expressing cells after spinal cord injury are glial progenitors

NG2(+) cells in the adult rat spinal cord proliferate after spinal cord injury (SCI) and are postulated to differentiate into mature glia to replace some of those lost to injury. To further study these putative endogenous precursors, tissue at 3 days after SCI or from uninjured adults was dissociated, myelin partially removed and replicate cultures grown in serum-containing or serum-free medium with or without growth factors for up to 7 days in vitro (DIV). Cell yield after SCI was 5-6 times higher than from the normal adult. Most cells were OX42(+) microglia/macrophages but there were also more than twice the normal number of NG2(+) cells. Most of these coexpressed A2B5 or nestin, as would be expected for glial progenitors. Few cells initially expressed mature astrocyte (GFAP) or oligodendrocyte (CC1) markers, but more did at 7 DIV, suggesting differentiation of glial precursors in vitro. To test the hypothesis that NG2(+) cells after SCI express progenitor-like properties, we prepared free-floating sphere and single cell cultures from purified suspension of NG2(+) cells from injured spinal cord. We found that sphere cultures could be passaged in free-floating subcultures, and upon attachment the spheres clonally derived from an acutely purified single cell differentiated into oligodendrocytes and rarely astrocytes. Taken together, these data support the hypothesis that SCI stimulates proliferation of NG2(+) cells that are glial progenitor cells. Better understanding the intrinsic properties of the NG2(+) cells stimulated by SCI may permit future therapeutic manipulations to improve recovery after SCI.     

Immunoreactivity for intracellular androgen receptors in identified subpopulations of neurons, astrocytes and oligodendrocytes in primate prefrontal cortex.

Sex differences in and hormone malleability of a variety of cognitive and mnemonic functions suggest that the association cortices in human and nonhuman primates are targets of gonadal hormone stimulation. One mechanism involved in this stimulation may be genomic actions mediated by intracellular androgen receptors. To identify potential cellular targets of this influence, single- and double-labeling immunohistochemical methods were used to precisely localize androgen receptor proteins in the prefrontal association cortex of adult rhesus monkeys. In both the dorsolateral and orbitofrontal regions, receptor antibodies labeled substantial populations of small intensely immunoreactive nuclei, as well as much larger and less strongly immunoreactive nuclei in all major cellular layers and/or in underlying white matter. Double-labeling studies revealed that large and small immunolabeled nuclei were further distinguished by colocalization with different classes of cell-specific markers. Whereas the large, pale receptor-immunoreactive nuclei colocalized with immunomarkers for neurons, the small, strongly immunoreactive nuclei colocalized exclusively with glial markers. Among androgen receptor-immunoreactive glia, a majority were immunoreactive for astrocyte markers, with smaller numbers of nuclei colocalized with oligodendrocyte markers; immunolabels for microglia failed to colocalize with androgen receptor immunoreactivity. This discovery of an unexpectedly large population of androgen receptor bearing glia suggests that direct functional interactions between endocrine signaling pathways and glial cells such as those coming into view in studies in subcortical and allocortical structures may also take place in the cerebral cortex and contribute to gonadal hormone stimulation of cortical processing of cognitive information.     

Radiation-induced apoptosis in the neonatal and adult rat spinal cord

This study was designed to characterize radiation-induced apoptosis in the spinal cord of the neonatal and young adult rat. Spinal cords (C2-T2) of 1-, 2- and 10-week-old rats were irradiated with a single dose of 8, 18 or 22 Gy. Apoptosis was assessed histologically according to its specific morphological features or by using the TUNEL assay. Cell proliferation was assessed immunohistochemically using BrdU. Identities of cell types undergoing apoptosis were assessed using immunohistochemistry or in situ hybridization using markers for neurons, glial progenitor cells, microglia, oligodendrocytes and astrocytes. The time course of radiation-induced apoptosis in 1- or 2-week-old rat spinal cord was similar to that in the young adult rat spinal cord. A peak response was observed at about 8 h after irradiation, and the apoptosis index returned to the levels in nonirradiated spinal cords at 24 h. The neonatal rat spinal cord demonstrated increased apoptosis compared to the adult. Values for total yield of apoptosis over 24 h induced by 8 Gy in the neonatal rat spinal cord were significantly greater than that in the adult. Immunohistochemistry studies using Leu7, galactocerebroside, Rip and adenomatous polyposis coli tumor suppressor protein indicated that most apoptotic cells were cells of the oligodendroglial lineage regardless of the age of the animal. No evidence of Gfap or factor VIII-related antigen-positive apoptotic cells was observed, and there was a small number of apoptotic microglial cells (lectin-Rca1 positive) in the neonatal and adult rat spinal cord. In the neonatal but not adult rat spinal cord, about 10% of the apoptotic cells appeared to be neurons and were immunoreactive for synaptophysin. Labeling indices (LI) for BrdU in nonirradiated 1- and 2-week-old rat spinal cord were 20.0 and 16.3%, respectively, significantly greater than the LI of 1.0% in the 10-week-old rat spinal cord. At 8 h after a single dose of 8 Gy, 13.4% of the apoptotic cells were BrdU-positive in 10-week-old rat spinal cord, whereas 62.4 and 44.1% of the apoptotic cells showed BrdU incorporation in 1- and 2-week-old rat spinal cord, respectively. Regardless of the age of the animal, the apoptosis indices in BrdU-positive cells were greater than those in BrdU-negative cells. We conclude that the neonatal spinal cord demonstrates a greater level of apoptosis after exposure to ionizing radiation than the young adult spinal cord. This increase in apoptosis may be associated in part with the greater percentage of proliferating cells in the neonatal spinal cord, which demonstrate a greater level of radiation-induced apoptosis than nonproliferating cells.     

Visualisation of specific binding sites for atrial natriuretic peptide on non-neuronal cells of cultured rat sympathetic ganglia

Summary The distribution of atrial natriuretic peptide binding sites on cells in dissociated culture preparations of neonatal rat superior cervical ganglia and in explant cultures of rat thoracic sympathetic chain ganglia has been studied. The autoradiographic visualisation of atrial natriuretic peptide binding sites has been combined with the use of specific immunocytochemical markers for glial cells (antiserum to S-100 protein), fibroblasts (antiserum to fibronectin) and neurones (antiserum to protein gene product 9.5) in order to achieve unambiguous identification of the cell types in culture. Specific binding sites for rat¹²⁵I-atrial natriuretic peptide_(1–28) were observed over subpopulations of fibronectin-like-immunoreactive fibroblasts and S-100-like-immunoreactive glia in the dissociated superior cervical ganglion cultures. However, only a subpopulation of fibronectin-like-immunoreactive fibroblasts possessed atrial natriuretic peptide binding sites in the explant culture preparations. No atrial natriuretic peptide-like-immunoreactive cells were present in either culture. The distribution of autoradiographic grains over individual cell surfaces in culture was uniform, but there were distinct differences in the density of labelling of single cells of the same type. This apparent variation in the number of binding sites on glial cells and fibroblasts in culture did not seem to be related to the morphology of the cells or the surrounding cell types. No sympathetic neurones were labelled with autoradiographic grains in either the dissociated or explant culture preparations. However, the presence of atrial natriuretic peptide binding sites on non-neuronal cells of sympathetic ganglia in culture may be linked to the relationship between atrial natriuretic peptide and the sympathetic nervous system.     

Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient.

The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.     

Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures

We describe the use of the neutral protease Dispase for the dissociation of neonatal rat brain tissue for the preparation of primary monolayer astrocyte cultures. The method involves 5 to 6 successive extractions with careful separation of sedimenting, undissociated tissue. This method gives an initial cell suspension of high viability (93.7±1.7% cells exclude trypan blue). In comparison trypsin (0.25%) dissociated tissue gave a cell suspension that showed a lower viability of 58.2±7.6%. Identical saturation densities of 1.1 to 1.2×10⁴ cells/cm² after two weeks in culture were obtained for a range of seeding densities from 1 to 4×10⁴ cells/cm² of the Dispase dissociated tissue. Staining for glial fibrillary acidic protein showed that 90–100% cells were positive for this astroglial marker. Thus, the use of Dispase for the initial dissociation of rat brain tissue seems to give primary astrocyte cultures which are very reproducible and homogeneous.     

Glial-cell-line-derived neurotrophic factor induces nerve fibre formation in primary cultures of adrenal chromaffin cells

Neurotrophic factors, such as nerve growth factor (NGF), have been shown to promote the differentiation of neural crest neuroblasts into sympathetic neurons, whereas glucocorticoids promote the endocrine phenotype of adrenal medullary chromaffin cells. This pluripotency is preserved to some extent in adult chromaffin cells, with NGF and other neurotrophic factors influencing the differentiation of these cells. In this study, the effects of glial cell line-derived neurotrophic factor (GDNF) on explanted chromaffin tissue have been investigated. The localization of mRNAs corresponding to the two components of the GDNF receptor, GDNF family receptor alpha 1 (GFRalpha1) and Ret, were demonstrated in adult adrenal medullary ganglion cells. GFRalpha1 mRNA was expressed in explanted chromaffin tissue at levels dependent on the presence of serum in the medium but decreased on the addition of blocking antibodies against transforming growth factor beta (TGFbeta). However, TGFbeta1 (1 ng/ml) did not upregulate GFRalpha1 mRNA expression when added to serum-free medium. GDNF induced neurite formation from chromaffin cells, as measured by the ratio of neurite-bearing versus total number of chromaffin cells in primary cultures of adult adrenal medulla. The most potent dose inducing neurites from chromaffin cells was 100 ng/ml GDNF. However, this dose was not as efficient as that seen when chromaffin cells were stimulated with NGF (100 ng/ml). Thus, adrenal medullary cells express mRNAs for the GDNF receptor components Ret and GFRalpha1, increase their expression upon being cultured in serum-containing medium and respond to GDNF treatment with an increase in the number of cells that develop nerve processes.     

Isolation and characterization of cells with neurogenic potential from adult skeletal muscle

Autologous cell therapies in neurodegenerative diseases and stroke will require an efficient generation of neuroprogenitors or neurons. We have previously shown that presumptive neural progenitors can be obtained from a candidate stem cell population isolated from adult skeletal muscle. Here we describe experimental conditions to isolate and characterize the cells with neurogenic potential from this population. Candidate stem cell population was isolated from adult skeletal muscle and expanded for selection during at least 30 cell divisions. FACS analysis revealed that this population was homogeneous with respect to CD45 (-), CD34 (-), and heterogeneous for CD90 (Thy-1) expression. The population was separated by cell sorting into three sub-populations based on CD90 expression (CD90-, CD90+, and CD90++) and each population expanded rapidly as free-floating spheres. When dissociated and plated in a neuronal differentiation medium, a large number of CD90+ cells acquired morphological characteristics of neuroprogenitors and neurons, and expressed markers of neurons but no markers of glial or muscle cells. In contrast, CD90- and CD90++ cells lacked this ability. Comparison of CD90+ and CD90- populations may be useful for studying the molecular characteristics defining the neuronal potential of stem cells from adult muscle. The selection of CD90+ expressing cells, combined with the growth conditions presented here, allows for rapid generation of a large number of cells which may be useful for autologous cell replacement therapies in the central nervous system.     

Detection and partial characterization of a developmentally regulated nuclear antigen in neural cells in vitro and in vivo

We report that a monoclonal antibody directed against phosphorylated neurofilaments (SMI 31) recognizes nuclear antigens present in embryonic but not in adult neural cells. On Western blots, the antibody reacts with four proteins of apparent MW 35, 37, 52/54, and 250 KD which are found exclusively in developing brain tissue. These nuclear antigens are expressed by glial and neuronal cells. Both nuclear staining and immunoreactive proteins decrease with ongoing in vitro differentiation. A computer search for proteins that share the epitope recognized by antibody SMI 31 did not yield any proteins of known nuclear localization that exhibit the same molecular weights and solubility characteristics as the above immunoreactive proteins. We conclude that antibody SMI 31 recognizes hitherto unknown nuclear proteins which, in neural cells, are developmentally regulated.     

Polydendrocytes display large lineage plasticity following focal cerebral ischemia

Polydendrocytes (also known as NG2 glial cells) constitute a fourth major glial cell type in the adult mammalian central nervous system (CNS) that is distinct from other cell types. Although much evidence suggests that these cells are multipotent in vitro, their differentiation potential in vivo under physiological or pathophysiological conditions is still controversial.To follow the fate of polydendrocytes after CNS pathology, permanent middle cerebral artery occlusion (MCAo), a commonly used model of focal cerebral ischemia, was carried out on adult NG2creBAC:ZEG double transgenic mice, in which enhanced green fluorescent protein (EGFP) is expressed in polydendrocytes and their progeny. The phenotype of the EGFP(+) cells was analyzed using immunohistochemistry and the patch-clamp technique 3, 7 and 14 days after MCAo. In sham-operated mice (control), EGFP(+) cells in the cortex expressed protein markers and displayed electrophysiological properties of polydendrocytes and oligodendrocytes. We did not detect any co-labeling of EGFP with neuronal, microglial or astroglial markers in this region, thus proving polydendrocyte unipotent differentiation potential under physiological conditions. Three days after MCAo the number of EGFP(+) cells in the gliotic tissue dramatically increased when compared to control animals, and these cells displayed properties of proliferating cells. However, in later phases after MCAo a large subpopulation of EGFP(+) cells expressed protein markers and electrophysiological properties of astrocytes that contribute to the formation of glial scar. Importantly, some EGFP(+) cells displayed membrane properties typical for neural precursor cells, and moreover these cells expressed doublecortin (DCX)--a marker of newly-derived neuronal cells. Taken together, our data indicate that polydendrocytes in the dorsal cortex display multipotent differentiation potential after focal ischemia.     

Immunocytochemical localization of an H2A variant in the spermatogenic cells of the mouse

Immunocytochemical localization of protein "A," an H2A variant, has been carried out in the adult, neonatal, and embryonic spermatogenic cells of the mouse using the peroxidase-antiperoxidase technique. The results indicate an apparent enrichment of this protein in the meiotic cells of the adult testis. In addition, T-prospermatogonia present in the neonatal mouse and 16-day-old embryos were found to be immunoreactive. By contrast, Sertoli cells and other somatic elements of the neonatal and embryonic gonads were only weakly immunoreactive. These data suggest potential usefulness of protein "A" as a nuclear marker of the embryonic spermatogenic cells.     

Smooth Muscle Strips for Intestinal Tissue Engineering

Functionally contracting smooth muscle is an essential part of the engineered intestine that has not been replicated in vitro. The purpose of this study is to produce contracting smooth muscle in culture by maintaining the native smooth muscle organization. We employed intact smooth muscle strips and compared them to dissociated smooth muscle cells in culture for 14 days. Cells isolated by enzymatic digestion quickly lost maturity markers for smooth muscle cells and contained few enteric neural and glial cells. Cultured smooth muscle strips exhibited periodic contraction and maintained neural and glial markers. Smooth muscle strips cultured for 14 days also exhibited regular fluctuation of intracellular calcium, whereas cultured smooth muscle cells did not. After implantation in omentum for 14 days on polycaprolactone scaffolds, smooth muscle strip constructs expressed high levels of smooth muscle maturity markers as well as enteric neural and glial cells. Intact smooth muscle strips may be a useful component for engineered intestinal smooth muscle.     

Glial cell growth in culture: Influence of living cell substrata

The role of the microenvironment in the growth of glial cells in culture has been the topic of ongoing research in this laboratory. Recently, we reported a study on the contribution of fibroblast cell substratum and extracellular matrix in glial cell growth. In the present study we report data concerning a) the influence of a neuronal-enriched living substratum from chick embryo on the growth of glial cells derived from chick embryonic brain and plated onto the substratum; b) the influence of dissociated cells derived from chick embryonic brain on the growth of established glial cells in culture, and c) the influence of dissociated cells derived from adult rat spinal cord on the growth of established glial cells from newborn rat in culture. The activities of glutamine synthetase (GS) and 2, 3-cyclic nucleotide 3-phosphohydrolase (CNP) were the biochemical probes determined for astrocytes and oligodendrocytes, respectively. We found that glial growth as assessed by both enzyme activities, was enhanced when a nervous tissue derived cell population was plated onto a glial-enriched substratum, whereas glial growth was inhibited when the neuronal-enriched population was the cell substratum.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.