Primary Cortical Cell Cultures Research Articles

Molecular neurobiological approach to the pathogenesis of epilepsy: a preliminary study

We studied the effects and the interactions of some candidate genes related to the pathogenesis of epilepsy using a domestic audiogenic epilepsy-prone rat, matched with the epilepsy-resistant Wistar rat, and primary fetal cerebral cortical neuronal cell cultures of both. The preliminary results showed that there was a possible abnormality of the CCK gene at the post-translational stage in the early postnatal period in P77PMC rat brain; the later rapidly increased rate of CCK-8 synthesis in the hippocampus and subcortical region may represent a compensatory response to the neuronal pathways involved in audiogenic seizures. CCK-8 can decrease the NMDA (1 microM)-induced free intracellular Ca2+ concentration, so it seems to be an inhibitory neuromodulator. In neuronal cell cultures, the NMDA (0.1 microM)-induced c-fos mRNA expression on culture day 18 in vitro was higher in P77PMC than Wistar rats (P < 0.05). Interleukin-1 (20 nM) can induce endogenous opioid mRNA expression and peptide release in neuronal cell cultures, which can be abolished by the addition of antisense oligos of c-fos/c-jun to cell cultures treated with interleukin-1. Both interleukin-1 and opioids can increase the free intracellular Ca2+ concentration, and their specific antagonists can reverse their effects, so they both seem to be excitatory neuromodulators in the CNS.

Assessment of cell-specific cytotoxic responses of the kidney to selected aromatic hydrocarbons

The present studies were conducted to assess cell-specific cytotoxic responses of the kidney to selected aromatic hydrocarbons (AHs) using in vitro culture systems of glomerular mesangial cells and cortical tubular epithelial cells. Primary cultures of glomerular mesangial cells or cortical tubular epithelial cells were treated for 24 hr with naphthalene (NAPH, 0.1–1000 μ m), 2-methylnaphthalene (2-MNAPH, 0.1–1000 μ m), benzo[ a]pyrene (B[ a]P, 0.3–300 μ m), pentachlorophenol (PCP, 0.1–1000 μ m), 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD, 0.001–1 n m) or 2,3,7,8-tetrachlorodibenzofuran (TCDF, 0.005–5 n m). [ 3H]Thymidine incorporation into DNA, cellular potassium content, and lactate dehydrogenase (LDH) leakage were used as indices of cytotoxicity. Glomerular mesangial cells exposed to NAPH or 2-MNAPH (0.1–1000 μ m) exhibited [ 3H]thymidine incorporation profiles similar to those of the vehicle controls, whereas B[ a]P at all concentrations and PCP at 1000 μ m decreased [ 3H]thymidine incorporation in comparison with the controls. TCDD and TCDF increased [ 3H]thymidine incorporation relative to controls at 0.1 n m and 0.5 n m, respectively. Glomerular mesangial cells treated with NAPH or PCP exhibited decreased cellular potassium content and increased LDH leakage only at the 1000 μ m concentration. In tubular epithelial cells, treatment with NAPH (10 or 1000 μ m), 2-MNAPH (10 or 1000 μ m), B[ a]P (300 μ m), and PCP (10 or 1000 μ m) induced concentration-dependent decreases in [ 3H]thymidine incorporation relative to controls. In contrast, exposure of tubular epithelial cells to TCDD or TCDF did not modulate [ 3H]thymidine incorporation at any of the concentrations tested. A modest decrease in cellular potassium content and an increase in LDH leakage was observed at the 1000 μ m concentration of PCP in tubular epithelial cells. On the basis of these data, we conclude that renal glomerular mesangial cells and tubular epithelial cells exhibit differential cytotoxic response profiles to AHs.

Renal cells transformed with SV40 contain a high-conductance calcium-insensitive potassium channel.

The inside-out variant of the patch-clamp technique was used to investigate the properties of a K+ channel occurring in 20% of patches from two renal cell lines transformed with the wild-type simian virus 40 (SV40) (Vandewalle et al. J. Cell. Physiol. 141: 203-221, 1989). This channel was practically absent from the primary cultures of renal cortical cells from which the cell lines were originally derived. With identical K(+)-rich solutions on both sides of the membrane patch, the channel showed an inwardly rectifying current-voltage relationship with unit conductances of 151.8 +/- 4.8 pS at negative and 86.4 +/- 5.9 pS at positive voltages (n = 18). When K+ in the bath was replaced by Na+, a mean reversal potential of 57.0 +/- 5.2 mV (n = 6) was observed from which a K(+)-to-Na+ permeability ratio of 13 was calculated. The channel was insensitive to internal Ca2+ and was blocked by internal Ba2+. No clear dependence on voltage was apparent. This channel bears no resemblance to any epithelial K+ channel and may be a novel type of K+ channel. Its occurrence in two transformed cell lines with quite distinct phenotypes, one of proximal cells (RC.SV1) and the other of thick ascending limb cells (RC.SV2), suggests that transformation by SV40 might be responsible for its appearance.

Stimulation of GABAergic neuron differentiation by NT-4/5 in cultures of rat cerebral cortex

Primary cultures of fetal rat cortical cells were used to investigate trophic effects of neurotrophin-4/5 (NT-4/5) on GABAergic neurons. Chronic administration of NT-4/5 resulted in a significant increase in high-affinity GABA uptake and in a stronger immunohistochemical staining pattern and bigger cell size of neurons visualized by GABA immunohistochemistry. Brain-derived neurotrophic factor (BDNF) exerted similar actions. These effects were observed in cultures prepared from rat fetuses from embryonic day (ED) 15 and ED18. The total number of cells was marginally increased in ED15 but not ED18 cultures. NT-4/5 did not elevate uptake of excitatory amino acids. Administration of NT-4/5 and BDNF resulted in higher levels of the calcium binding protein, calbindin, in these cultures. Our findings suggest that subpopulations of cortical GABAergic and calbindin-expressing neurons respond to NT-4/5 during development.

Transepithelial phosphate transport in rabbit proximal tubular cells adapted to phosphate deprivation.

Both renal and nonrenal cells in culture adapt to deprivation of Pi by increasing Na-dependent Pi uptake. We studied whether this change in uptake is reflected in an increased renal transepithelial Pi transport. We grew primary cultures of rabbit renal cortical cells in plastic flasks and subcultured them onto Millicell-HA filters. This produced cell monolayers, which structurally and functionally resembled proximal tubule. These cells performed Na-dependent net transepithelial transport of 32Pi in the apical-to-basolateral direction that was inhibited by phosphonoformic acid in the apical fluid or by ouabain in the basolateral fluid or by preincubation with parathyroid hormone. Overnight incubation at low Pi concentrations led to a progressive increase in 5-min Na-dependent Pi uptake into cell monolayers. Na-dependent Pi uptake was threefold higher following overnight incubation at 25 microM Pi, compared with 3 mM Pi, and the increase was one-half maximal with incubation at an extracellular Pi concentration ([Pi]) of 300 microM. This was associated with a decrease in Na-dependent transepithelial Pi flux to the basolateral fluid by the same cells, which fell dramatically following incubation at < or = 300 microM Pi. There was no change in Na-dependent uptake or transepithelial transport of L-glutamine. This adaptation to Pi deprivation in vitro appears to serve to restore depleted cell stores of Pi rather than to regulate transepithelial Pi transport.

Rapid Communication Isolation and Characterization of the Promoter of the Human GABAA Receptor α1 Subunit Gene

The GABAA receptor, as assessed by ligand binding and chloride flux measurement in vivo and in vitro, is down-regulated in response to chronic benzodiazepine exposure. The mRNA levels of the alpha 1 and gamma 2 subunits of the receptor are also reduced. We have isolated the promoter of the gene encoding the alpha 1 subunit of the GABAA receptor to elucidate the regulatory mechanism of its expression. A DNA segment 650 bp long has been isolated that includes 151 bp of untranslated 5' end of the cDNA sequence and 500 bp of potential promoter-enhancer region. The transcriptional activity of this DNA segment linked to the firefly luciferase gene showed a strong orientation specificity. The promoter activity was localized to a 60-bp segment by deletion mapping. Mobility shift binding assay results suggest that this segment may interact with one or more factors in HeLa cell nuclear extracts to form a transcriptional complex. Primary cultures of embryonic chick cortical cells transfected with the promoter-luciferase construct were treated chronically with lorazepam. Transcriptional activity of this promoter construct was strongly repressed by chronic administration of lorazepam.

Formation and maturation of synapses in primary cultures of cerebral cortical cells

Formation and maturation of synapses in primary cultures of cerebral cortical cells

A digitized fluorescence imaging study of intracellular free calcium, mitochondrial integrity and cytotoxicity in rat renal cells exposed to ionomycin, a calcium ionophore

The objective of this study was to explore the role of extracellular Ca 2+ and mitochondrial integrity in ionomycin-induced cytotoxicity in primary cultures of rat kidney cortical epithelial cells using dignitized fluorescence imaging (DFI), which is a powerful tool for continuously observing the dynamic intracellular biochemistry of single living cells. Using DFI, intracellular free calcium ion concentration ([Ca 2+] i), mitochondrial membrane potential and loss of cell viability in individual rat renal cortical epithelial cells were examined temporally by fura-2, rhodamine 123 (Rh-123) and propidium iodide (PI), respectively. Images were taken within 10 min after exposure to 5 and 10 μM ionomycin. These three parameters, [Ca 2+] i, mitochondrial membrane potential and cell viability, were also measured in populations of cells by a multiwell fluorescence scanner with fluo-3, Rh-123 and PI, respectively. Cytotoxicity was also assessed by two colorimetric cytotoxicity tests (LDH leakage and mitochondrial MTT reduction). Using DFI, the fluorescence scanner and the colorimetric cytotoxicity tests, we found that exposure of primary cultures of rat kidney cortical epithelial cells to high concentrations of ionomycin (5 and 10 μM) caused a rapid and sustained rise in [Ca 2+] i, which preceded dissipation of the mitochondrial membrane potential and loss of cell viability and that chelation of extracellular Ca 2+ with EGTA attenuated these responses. We demonstrated the value of using DFI to continuously observe the dynamic intracellular biochemistry of single living cells by establishing a sequence of elevated [Ca 2+] i, dissipation of mitochondrial membrane potential and cytotoxicity. We conclude that a combination of the influx of extracellular Ca 2+ and loss of mitochondrial integrity may be responsible for the cytotoxicity observed in individual renal cells and populations of renal cells after treatment with ionomycin.

Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl- d-aspartate neurotoxicity

We examined the role of nitric oxide in N-methyl- d-aspartate (NMDA) receptor-mediated neurotoxicity in rat and mouse primary cortical cell cultures. In rat and mouse cultures, the NO synthase inhibitor, N G-Nitro- l-arginine, blocked cGMP formation but not neuronal cell death following a 5–10 min exposure to 300–500 μM NMDA. N G-Monomethyl- l-arginine was also unable to prevent neuronal death. In contrast, the non-competitive NMDA receptor antagonist, dextrophan, prevented both cGMP formation and cell death. While other data suggest that the synthesis of nitric oxide can mediate NMDA receptor-mediated neurotoxicity, present results suggest that such synthesis is not necessarily required.

Cadmium Uptake by Primary Cultures of Rat Renal Cortical Epithelial Cells: Influence of Cell Density and Other Metal Ions

The uptake and accumulation of 1-5 μM cadmium (Cd) was studied in primary cultures of rat renal cortical epithelial cells under protein-free conditions at 4 or 37°C for up to 30 min. The cells were isolated from female rats by collagenase digestion and cultured for 3-7 days. Confluency of the culture, monitored morphologically as well as by total protein content, was achieved on Day 5. Cd accumulation at 1 μM concentration demonstrated an inverse relationship to the cell density; the Cd level in Day 5 culture was only 45% of that in Day 3 culture. In subconfluent cultures (Days 3 and 4) the Cd accumulation was temperature sensitive; on Day 3 the cells accumulated one-third less Cd at 4°C than at 37°C. In comparison, the confluent cells (Day 5) had the same Cd accumulation regardless of the incubation temperature. In these cells, preincubation with cyanide also had no significant effect on Cd accumulation, implying a lack of energy requirement for Cd uptake. As the transport of Cd may involve processes that exist for the essential metal ions, the effect of 30 μM zinc (Zn) and copper (Cu) on the accumulation of 1 μM Cd was studied in Day 5 cultures. Coincubation with Zn caused a 16% reduction in Cd levels at 37°C and even greater reduction (44% of control) at 4°C. Similarly, Cu inhibited Cd accumulation by 26 and 45% at 37 and 4°C, respectively, as compared to the temperature-matched controls. The V max for the initial Cd uptake (1 min) was 125 pmol/mg protein/mm and the K m was 7 μM. Both Zn and Cu exhibited competitive inhibition kinetics and doubled the K m for Cd uptake. The K i for Zn and Cu was 23 and 30 μM, respectively. Mercury (Hg) and lead (Pb) were also tested for their ability to affect Cd accumulation. As compared to the controls, 1 μM Hg caused an 11% reduction in Cd level at 37°C. In contrast, 1 μM Pb enhanced Cd accumulation by 20%. However, neither Hg nor Pb had any significant effect on Cd accumulation at 4°C. All four metals had no significant effect on the efflux of Cd from the cells. Thus, these metals affected Cd accumulation by changing its uptake rather than its efflux. The results of Cd accumulation at varying cell densities demonstrated that as the proximal renal epithelial cells became confluent, and supposedly formed tight junctions and developed brush border, the Cd uptake became more restricted than under subconfluent culture conditions where the uptake was apparently through the basolateral membrane. Lack of effect of cyanide and lower temperature on Cd accumulation in confluent cultures further suggests that Cd transport through the apical membrane might occur by diffusion. However, under subconfluent culture conditions, where Cd accumulation is temperature sensitive, the transport through the basolateral membrane could possibly occur via ion channels or carriers.

Mitogenic action, regulation, and localization of insulin-like growth factors in the human fetal adrenal gland.

Polypeptide growth factors may play an important role in the regulation of human fetal adrenal cortical growth by mediating the tropic actions of ACTH. The abundance of mRNA encoding insulin-like growth factor-II (IGF-II) is high in the human fetal adrenal gland and is stimulated by ACTH in cultured fetal adrenal cortical cells. Therefore, we studied the mitogenic action, regulation, and localization of IGF-II and a closely related peptide, IGF-I, in primary cultures of human fetal adrenal cortical cells and whole human fetal adrenal glands. Recombinant human IGF-I and IGF-II stimulated proliferation of fetal adrenal cortical cells in a dose-dependent fashion (1-1000 ng/mL; 0.133-133 nM). At 1000 ng/mL (133 nM), both peptides increased cell number 1.8- to 2-fold. Combinations of IGF-I or -II (100 ng/mL; 13.3 nM) with basic fibroblast growth factor (bFGF; 0.1 ng/mL; 6 pM) or epidermal growth factor (EGF; 1.0 ng/mL; 0.17 nM) had a greater effect on proliferation than bFGF, EGF, or either of the IGFs alone, suggesting an additive interaction. IGF-II mRNA was detected in cultured adrenal cortical cells by in situ hybridization analysis, and its abundance was stimulated by ACTH. In contrast, IGF-I mRNA was not detected in cultured fetal zone cells and was not regulated by ACTH. In whole human fetal adrenal glands, IGF-II mRNA was detected in the definitive and fetal zones and in the capsule, whereas IGF-I mRNA was detected only in the capsule and not in the two cortical zones. Using Northern blot analysis, we found that mRNA encoding IGF-II was present in high abundance in fetal adrenal glands (16-22 weeks) and barely detectable in adult adrenals, whereas mRNA encoding IGF-I was present in very low abundance in the fetal adrenal, but was high in adult human adrenals. As IGF-II expression is high in the human fetal adrenal cortex and is regulated by ACTH, we propose that it is the dominant of the two IGFs regulating human fetal adrenal growth. The cooperative mitogenic effect of IGF-II with bFGF and EGF and the regulation of its expression by ACTH support the hypothesis that IGF-II may act as a mediator, in concert with bFGF and possibly EGF, of the tropic action of ACTH in regulating the rapid growth of the human fetal adrenal cortex during midgestation.

Formation and maturation of synapses in primary cultures of rat cerebral cortical cells: an electron microscopic study

Cerebral cortical cells from fetal rats (18 days) were cultured for 3, 7, 14, 21, 28, 35 days in vitro (DIV) and the development of synapses was examined morphologically by electron microscopy. At 3 DIV, no synapses could be recognized. An immature type of synapse was found at 7 DIV which thereafter developed morphologically. The length of the synaptic contact zone (SCZ) increased with DIV from 271 ± 11.4 nm (mean ± SEM) at 7 DIV to 410 ± 11.4 nm at 35 DIV. The number of synaptic vesicles per terminal also increased with DIV: 10.0 ± 1.2 at 7 DIV, 35.7 ± 3.4 at 21 DIV, and 53.3 ± 4.5 at 35 DIV. The time course of numerical density of synapses was examined quantitatively by electron microscopy. Synaptic density was very low at 7 DIV. It was significantly increased at 14 DIV and thereafter showed variable changes. Four culture series showed decreases after 14 DIV, but one series showed a further increase at 21 DIV followed by a decrease at 28 DIV. The mean density of synapses at each DIV was as follows: 1780 ± 86 10 6 μ m 3 at 7 DIV, 4244 ± 595 10 6 μ m 3 at 14 DIV, 2285 ± 674 10 6 μ m 3 at 21 DIV, 2552 ± 646 10 6 μ m 3 at 28 DIV, and 2080 ± 532 10 6 μ m 3 at 35 DIV. Neuronal cell density was counted in each culture to calculate the relative number of synapses per neuron. The cell density decreased with age from 301 ± 51 10 6 μ m 3 at 7 DIV to 39 ± 9 10 6 μ m 3 at 35 DIV. The number of synapse per neuron increased with DIV to the end of the 4th week (from 64.1 ± 9.8 at 7 DIV to 1130.1 ± 254.3 at 28 DIV). The time course of synapse formation in culture resembled that in the cerebral cortex in vivo.

Nitric oxide synthase induction in glial cells: Effect on neuronal survival

In primary rat cortical glial cell cultures lipopolysaccharide (LPS) induced a dose- and time-dependent increase of intracellular cyclic GMP concentration associated with a release of nitrite. The LPS-induced cyclic GMP and nitrite increase was enhanced by interferon-γ and was prevented by L-N G- nitroarginine, dexamethasone and cycloheximide. Thus indicates that LPS effect occured via the production of nitric oxide (NO) and involved new protein synthesis suggesting the induction of NO syntahse in these cells. Furthermore this induction was Ca 2+-independent and was blocked by an inhibitor of the synthesis of tetrahydrobiopterin. The inducible NO synthase was also expressed by C6 glioma cells. In primary mixed cultures containing both neuronal and glial cells, the effects of LPS were less important than in primary glial cell cultures suggesting that glial cells rather than neurons expressed the inducible form of NO synthase. On the other hand no change on neuronal viability was observed after NO synthase induction by LPS in this culture type. This study indicates that glial cells are able to induce NO synthase without affecting neuronal survival.

Comparative toxicity and accumulation of cadmium chloride and cadmium-metallothionein in primary cells and cell lines of rat intestine, liver and kidney

The protective role of metallothionein (Mt) in the toxicity of cadmium (Cd) is controversial, since Cd bound to Mt is more nephrotoxic than ionic Cd after parenteral exposure and less hepatotoxic than ionic Cd after oral exposure. This study compared the uptake and toxicity in vitro of CdCl 2 and two isoforms of rat cadmium-metallothionein (CdMt-1 and CdMt-2) using primary rat kidney cortex cells, primary rat hepatocytes, liver hepatoma cell line H-35, kidney epithelial cell line NRK52-E and intestinal epithelial cell line IEC-18. The molar ratio of Cd was 2.1 and 1.4 mol Cd/mol Mt for CdMt-1 and CdMt-2, respectively. Monolayer cultures were incubated for 22 hr with CdCl 2, CdMt-1 or CdMt-2 and Cd accumulation was examined at Cd levels of 0.25–10 μM-Cd. Cells exposed to CdCl 2 accumulated more Cd in 22 hr than cells exposed to an equimolar amount of CdMt. For CdCl 2 the Cd accumulation is directly related to the Cd concentration in the medium; however, for CdMt an increase in Cd concentration in the medium above 2 μM had no effect on the Cd accumulation in the cells. At Cd concentrations above 2 μM, therefore, the difference in Cd accumulation between CdCl 2 and CdMt was greater (5–6 times) than at concentrations below 2 μM (1–2 times). Cytotoxicity was examined in the Cd-concentration range from 0.25 to 100 μM by determining the lactate dehydrogenase (LDH) release in the medium and the neutral red uptake in the cells. Under these culture conditions CdCl 2 was at least 100 times more toxic than CdMt-1 or CdMt-2 in all cell types tested. Primary hepatocyte cultures were 10 times more sensitive (50% LDH release at 1–2 μM) to CdCl 2 intoxication than primary cultures of renal cortical cells or the intestinal cell line (50% LDH release at 10–20 μM). Hepatic and renal cell lines were less sensitive (50% LDH release at 20–35 μM) than the corresponding primary cultures. No difference in sensitivity towards CdMt-1 or CdMt-2 was found for the various cell types tested. To investigate the influence of the molar Cd ratio of CdMt on cytotoxicity, the Cd content of CdMt-1 (2.1 mol Cd/mol Mt) was artificially raised in vitro to 5 mol/mol Mt. Compared with native CdMt, CdMt with a high molar Cd ratio in primary renal cultures showed a 15% increase in LDH release at a Cd concentration of 1500 μM in the medium. In conclusion, exogenous CdMt is far less toxic than CdCl 2 to cell cultures in a serum-free medium. Whereas CdCl 2 in all cases showed dose-dependent Cd accumulation, Cd accumulation due to CdMt exposure in all cell types tested reached a plateau at medium Cd concentrations of 2 μM. The low cellular Cd uptake of CdMt and the corresponding low cytotoxicity supports previously reported results in vivo, showing that the difference in toxicity between CdMt and CdCl 2 is associated with a difference in Cd distribution.

The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin.

Urine contains proteins that inhibit the growth of calcium oxalate (CaOx) crystals and may prevent the formation of kidney stones. We have identified a potent crystal growth inhibitor in the conditioned media from primary cultures of mouse kidney cortical cells. Conditioned media, incubated with the kidney cells for 6-72 h, was assayed for crystal growth inhibition; inhibitory activity increased 15-fold by 24 h. Inhibitory activity was purified from serum-free media containing proteinase inhibitors using anion-exchange and gel-filtration chromatography. A single band of molecular weight 80,000 daltons was seen after SDS-polyacrylamide gel electrophoresis. The sequence of the N-terminal 21 amino acids of this protein matched that of osteopontin (OP), a phosphoprotein initially isolated from bone matrix. Antisera raised to fusion proteins produced by plasmids containing the N-terminal or C-terminal portions of OP cDNA also cross-reacted with the protein purified from cell culture media on western blots. The effect of the purified protein on the growth of CaOx crystals was measured using a constant composition assay. A 50% inhibition of growth occurred at a protein concentration of 0.85 micrograms/ml, and the dissociation constant of the protein with respect to CaOx crystal was 3.7 x 10(-8) M. The concentration of OP in mouse urine, measured using antibodies raised to the purified protein, was approximately 8 micrograms/ml. We conclude that OP is synthesized by kidney cortical tubule cells and functions as a crystal growth inhibitory protein in urine.

Dynamic features of cells expressing macrophage properties in tissue cultures of dissociated cerebral cortex from the rat

SummaryTwo previously identified forms of macrophage were investigated in primary cultures of cerebral cortical cells. Dynamic features were revealed through time-lapse video recording and aspects of macrophage function were assessed. The two cell forms were shown to be different pre-mitotic stages of a single cell type. The cell cycle for these cells involved an initial large, flat, quiescent cell which retracted to yield a slightly rounded form with numerous processes. This latter form lost processes and developed profuse filopodia as it became very rounded just prior to division; both resulting daughter cells then regained the initial large flat appearance. These cells possessed several properties of macrophages, including phagocytosis, nucleoside diphosphatase enzyme, and CR3 receptors. These properties were transient, expressed just before and after mitosis, but subsequently down-regulated in the flat daughter cells. Because of this feature, it was difficult to determine the exact size of this cell population; however, the observed rate of proliferation suggests it may be substantial. It is suggested that these cells correspond to non-microglial macrophages of brain tissue and, because of their significant down-regulation, they may be difficult to detect. This may be important in studies of brain accessory immune cells in tissue culture.

Pinocytosis as a select marker of ramified microglia in vivo and in vitro.

Based on previous observations in tissue culture, we investigated pinocytotic activity as a potential cell marker for brain microglia. This functional activity was assessed in three different preparations derived from rat: primary cultures of mixed cerebral cortical cells, tissue slabs of whole cerebrum, and cultures of isolated or enriched microglial cells. Each preparation was incubated with the fluorescent dye lucifer yellow as a soluble tracer and then processed for light microscopy. Under the conditions utilized, ramified microglia specifically exhibited differentially high pinocytotic labeling in all cases; the dye was mainly localized within the cell somata, where it was sequestered in pinocytotic vesicles. In each preparation, the identity of the labeled cell population was confirmed as microglia through immunohistochemical staining with the monoclonal antibody (MAb) OX-42, a specific microglial marker. Therefore, pinocytotic labeling is proposed as a select cell marker for microglia, which may be extremely useful in the identification and study of ramified microglial cells.

Characterization of ramified microglia in tissue culture: Pinocytosis and motility

Functional properties of ramified microglia were investigated in primary cultures of rat cerebral cortical cells. These microglia could be readily identified in both fixed and living cultures through previously established features. Based on their destruction by 5 mM L-leucine methyl ester, a high level of intrinsic endocytotic activity was established. When cultures were incubated with fluorescent latex beads to assess phagocytosis, little or no such activity was exhibited by ramified cells. However, when cultures were incubated with dyes or other soluble tracer compounds, these cells always exhibited labeling. This labeling was selective for ramified microglia in the cultures and was demonstrated using a variety of compounds, including trypan blue, lucifer yellow, horseradish peroxidase (HRP), and India ink. Intracellular label could be observed in vesicular structures; this localization corresponded to an active cellular process. Also, cellular labeling was inhibited by the presence of colchicine. These features supported the inference that the labeling was attributable to pinocytosis, and this process appeared to account for the vast majority of endocytotic activity in the ramified microglia. Possible physiological significance of this pinocytotic activity was indicated by the accumulation of various neurotransmitters/modulators: gamma-aminobutyric acid and vasoactive intestinal polypeptide (VIP). Ramified cells in these cultures have been previously noted to exhibit a constant and rapid pattern of motility, which was consistently observed here through time-lapse video recording; pinocytosis and rapid motility were shown to concur in individual cells. Based on their high intrinsic pinocytotic activity and pattern of cellular motility, the ramified microglia specifically are suggested to serve a constitutive function of fluid cleansing within the interstitial spaces of brain tissue.

Lack of Changes in Cytosolic Ionized Calcium in Primary Cultures of Rat Kidney Cortical Cells Exposed to Cytotoxic Concentrations of Gentamicin: A Fluorescent Digital Imaging Method for Assessing Changes in Cytosolic Ionized Calcium

Digital imaging of cytosolic ionized calcium ([Ca2+]i) is a new technology made possible by the advent of newer fluorescent probes for [Ca2+]i and computerized imaging. A redistribution of cytosolic calcium has been suggested as a possible mechanism of the cytotoxicity of gentamicin; however, this hypothesis has never been tested. The present study employed digital imaging to determine whether a redistribution of cytosolic calcium preceded or accompanied cytotoxic exposures of primary rat renal cortical epithelial cultures to gentamicin. Concentrations of gentamicin and durations of exposure were chosen that precede and accompany significant cell death, as measured by the release of cytosolic lactate dehydrogenase (LDH). We have previously shown that no abrupt elevation of [Ca2+]i follows exposure to high concentrations of gentamicin. In the present study, no redistribution of cytosolic calcium was observed. However, several cellular reactions strongly demonstrated the normal uptake and intracellular proc...

Failure of gentamicin to elevate cellular malondialdehyde content or increase generation of intracellular reactive oxygen species in primary cultures of renal cortical epithelial cells

The role of lipid peroxidation in gentamicin cytotoxicity was assessed in primary cultures of renal cortical epithelial cells. The cellular content of malondialdehyde (MDA), one of the end products of lipid peroxidation, was determined, and the intracellular generation of reactive oxygen species, one of the events commonly occurring at the beginning of the process of lipid peroxidation, was estimated. Exposures to gentamicin were chosen that preceded or accompanied overt toxicity. MDA was determined by the thiobarbituric acid reactive substances assay. Intracellular generation of reactive oxygen species was estimated by quantitating the fluorescence of 2′,7′-dichlorofluorescein (DCF'), a fluorophore formed by the reaction of the deacetylation product (2′,7′-dichlorofluorescin, DCF) of the fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFDA) with reactive oxygen species. Neither elevated MDA content nor intracellular generation of reactive oxygen species was observed in primary cultures of renal cortical epithelial cells treated with gentamicin, using exposures which preceded or accompanied overt toxicity. We conclude that lipid peroxidation does not play a role in gentamicin cytotoxicity.