Biomedical subjects

W Cammer

Publications and source records attributed to W Cammer.

At least 19 recordsLinked to original sources

PubMed2001

Structural dynamics of oligodendrocyte lysis by perforin in culture: relevance to multiple sclerosis.

The mechanism by which oligodendrocytes are depleted from active lesions in multiple sclerosis (MS) is not clear but many reports implicate a cytolytic process. The most applied animal model for MS, chronic relapsing experimental autoimmune encephalomyelitis (EAE), has been established in inbred strains of mice, especially SJL and PL. Studies on oligodendrocytes from these strains in vitro have been hampered to date by an inability to grow these cells from mouse CNS tissue. We report here a successful method to culture SJL mouse oligodendrocytes and have analyzed lysis of these cells in vitro mediated by the pore-forming protein, perforin, a candidate effector molecule in inflammatory demyelination. Cultures were exposed to murine perforin, 36-72 hemolytic U, for up to 2.5 hr and examined using the oligodendrocyte phenotypic markers O4, galactocerebroside and myelin basic protein (MBP), in addition to a membrane dye (DiI) and a marker of necrosis, propidium iodide, (PI). Cultures were imaged chronologically by phase contrast, immunofluorescence, digital, light and electron microscopy. Findings showed that the majority of oligodendrocytes were killed within 60-90 min via pore expansion and ultimately, membrane disruption. The structural features of the cellular damage comprised swelling of the cell body, fenestration and fragmentation of membranes and processes, cytoplasmic vacuolation and breakdown of the nuclear envelope. Astrocytes in the same system were relatively resistant to cell lysis. The above patterns of oligodendrocyte damage in SJL oligodendrocytes were reminiscent of patterns in the MS lesion, leaving us to conclude that perforin may play an important role in the human disease.

Animals↗

PubMed2001

Oligodendrocyte killing by quinolinic acid in vitro.

Quinolinic acid, which is produced by macrophages and microglia, can kill neurons in vivo and in vitro. To test whether quinolinic acid is toxic to oligodendrocytes, glial cells cultured from the brains of 2-day-old rats were incubated with quinolinic acid at concentrations known to kill neurons. The cells were then fixed and immunostained with MAbO4 to mark immature and mature oligodendrocytes and anti-myelin basic protein (MBP) to mark mature oligodendrocytes. The data indicated up to 54% reductions in the numbers of O4-positive cells in cultures after incubation with quinolinic acid. Apoptosis of O4-positive cells began during the first 6 h, and some of the apoptotic cells became fragmented. Further apoptosis, and clumping of dead MBP-positive oligodendrocytes, occurred during longer incubation with quinolinic acid. Thus, quinolinic acid arising from macrophages and microglia during autoimmune disease may take part in a mechanism of oligodendrocyte injury and killing.

Animals↗

PubMed2000

Effects of TNFalpha on immature and mature oligodendrocytes and their progenitors in vitro.

Tumor necrosis factor alpha (TNFalpha) appears to take part in the pathogenesis of multiple sclerosis and to contribute to the degeneration of oligodendrocytes as well as neurons. TNFalpha is produced by microglia and astrocytes, which also produce hormones and cytokines that influence its biological activity. Thus, in mixed cultures the effects of exogenous TNFalpha might be modified by products of astrocytes and microglia. The effects of TNFalpha in oligodendrocyte-enriched cultures are reported below. We prepared the cultures by shaking oligodendrocytes off primary mixed glial-cell cultures from brains of 2-day-old rats at 7 days in vitro and plating them (0 days post-shake, DPS). Platelet-derived growth factor and fibroblast growth factor were included in the media at 1-5 DPS in order to encourage proliferation. At 2 DPS media were added with no TNFalpha (controls) or 1000, 2000 or 5000 U/ml of TNFalpha, and at 5 DPS media were replaced with fresh serum-free media. Cultures were fixed with 4% paraformaldehyde at 5, 7, 9 and 12 DPS and immunostained. Oligodendrocyte progenitors were not reduced in numbers immediately after the incubation with TNFalpha (i. e. at 5 DPS). However, after an additional 4 days in culture fewer progenitors remained in the cultures that had been treated with TNFalpha than in the untreated cultures. In the absence of the growth factors there were fewer progenitors, but their numbers also were reduced by TNFalpha. Maturation to the myelin basic protein (MBP)-positive stage was inhibited by about 36% at 9 DPS by 1000-2000 U/ml of TNFalpha, while numbers of O4+/MBP- precursors were unaffected. It is interesting that the steady-state number of O4-positive precursors was unchanged by TNFalpha at 9 DPS, when there were reductions in the numbers of A2B5-positive progenitors and MBP-positive mature oligodendrocytes. That observation suggests that the rates of proliferation, death and maturation are controlled by multiple factors, with a particularly vulnerable time at the maturation to the MBP-positive stage. At 5000 U/ml TNFalpha the specific effect on maturation was overtaken cytotoxicity. These data and a summary of the literature suggest that inhibition of MBP expression is sensitive to lower TNFalpha concentrations and incubation times than is cell survival. Specific effects on numbers of MBP-positive cells, morphology and MBP expression occur at 1000-2000 U/ml for 48-72 h or at up to 10000 U/ml for</=24 h, and the deficits remain after removal of the TNFalpha.

Animals↗

PubMed2000

Activity-dependent pH shifts in hippocampal slices from normal and carbonic anhydrase II-deficient mice.

The type II isoform of carbonic anhydrase is abundant in astrocytes and oligodendroglia. To explore whether the expression of the type II isoform is required for interstitial carbonic anhydrase activity, we studied extracellular pH transients in hippocampal slices from mutant mice devoid of carbonic anhydrase type II and from wild-type littermates. Stimulation of the Schaffer collateral afferents evoked similar extracellular pH transients in the CA1 stratum pyramidale, consisting of a predominant alkaline shift and little or no subsequent acidosis. After 5-s stimulus trains at 10 Hz, alkaline shifts were not significantly different in carbonic anhydrase II-deficient and wild-type preparations, averaging 0.09 +/- 0.04 and 0.08 +/- 0.04 unit pH, respectively. Addition of 1.5 microM benzolamide amplified the alkaline shifts by 385 +/- 146 and 345 +/- 75% in the mutant and wild-type preparations, respectively. Dose response studies with benzolamide displayed similar sensitivity to this carbonic anhydrase inhibitor over a concentration range of 0. 03-10 microM. These data indicate that interstitial carbonic anhydrase activity is effectively unaltered in brains devoid of carbonic anhydrase type II. The results are consistent with the interpretation that a distinct extracellular isoform of carbonic anhydrase exists in brain.

Action Potentials↗

PubMed1999

The neurotoxicant, cuprizone, retards the differentiation of oligodendrocytes in vitro.

The effects of oxalyldihydrazone (cuprizone) on weanling rodents provided an early protocol for toxic demyelination in vivo, in which degeneration of oligodendrocytes preceded disruption of the myelin sheath, and in which remyelination could take place. We administered cuprizone to oligodendrocyte-enriched glial-cell cultures and to mixed glial-cell cultures from neonatal rat brains. The cultures were treated with cuprizone for 1 h and allowed to continue differentiating on subsequent days. Treated cultures and respective control cultures were fixed with 4% paraformaldehyde (w/v) and immunostained with double immunofluorescence. MAbO4 was used to mark precursors and mature oligodendrocytes, and anti-myelin basic protein (MBP) to mark mature oligodendrocytes (O4+/MBP+), as distinguished from precursors, which were O4+/MBP-. Cell counts suggested that cuprizone inhibited the maturation of oligodendrocytes without diminishing the numbers of precursors, and appeared to affect the mitochondria in those cells.

Acridine Orange↗

PubMed1999

Atypical glial cells in demyelinated and hypomyelinated mouse brains.

Bis-cyclohexanone oxalyldihydrazone (cuprizone) was administered to young adult mice in order to investigate the hypothesis that the differentiation of oligodendrocyte progenitors or precursors, or of immature oligodendrocytes, might be interrupted during cuprizone intoxication. Vibratome sections were prepared from brains from control mice, mice that were fed cuprizone for 27 days and mice that were fed cuprizone for 25 days, followed by normal diet for 2 days, and the sections were immunostained with monoclonal antibodies: MAbO4, which is directed against galactocerebroside sulfate (sulfatide); and RMAb, which is directed against galactocerebroside (GC). Process-bearing RMAb+/O4- cells were abundant in the brains of mice that had consumed cuprizone for 27 days, and the numbers of O4-positive cells were subnormal. Two days after refeeding the normal diet the RMAb+/O4- structures were less abundant and O4-positive cells more numerous. Moreover, the numbers of O4-positive cells were only approximately 20% of normal in the brains of hypomyelinating jimpy mutant mice, while the numbers of RMAb-positive cells were approximately 80% of normal, and the processes of the latter were associated with axons. It is suggested that RMAb+/O4- cells in the affected brains may be process-bearing oligodendrocyte precursors containing unsulfated GC or a toxic galactolipid.

Animals↗

PubMed1999

Maturation of oligodendrocytes is more sensitive to TNF alpha than is survival of precursors and immature oligodendrocytes.

TNF alpha is a cytokine recently found at high levels in multiple sclerosis plaques. The present study addressed the questions whether TNF alpha might affect oligodendrocytes at various stages of maturation and whether the effects of transient incubation with TNF alpha would last during subsequent differentiation. Primary glial-cell cultures were treated with 1000 U/ml of TNF alpha for 48 h, beginning on days 1, 2, 6, 8 and 10 days in vitro, and allowed to grow for up to 3 weeks (total) in vitro. A significant deficit of O4+/MBP+ cells in the TNF alpha-treated cultures became obvious during the second week. Moreover, the morphology of the O4-positive cells became more complex with time in the control cultures, whereas fewer cells in TNF alpha-treated cultures developed into cells with sheets of membrane or > four processes. In TNF alpha-treated cultures, the numbers of O4-positive cells increased by about four-fold during weeks 2 and 3, but the numbers of MBP-positive cells did not and were significantly lower than the numbers of MBP-positive cells in control cultures. The effects of TNF alpha were apparent 1 to 14 days after treatment, suggesting long-term influences, and could be initiated at diverse stages of maturation. Future testing of hypothetical mechanisms by which TNF alpha may inhibit oligodendrocyte differentiation should impact on our understanding of the apparent limitations on remyelination in the mature CNS.

Animals↗

PubMed1999

Transplantation of glial cell progenitors from brains of normal rats and mice into brains of neonatal carbonic anhydrase II-deficient mutant mice.

Carbonic anhydrase II (CAII) is expressed in oligodendrocytes, astrocytes and myelin in brains of rats and normal mice, but not in the brains of CAII-deficient mutant mice. We have transplanted mixed glial-cell suspensions from normal mouse brains, and oligodendrocyte-enriched precursor cells cultured from normal rat brains, respectively, into the brains of neonatal CAII-deficient mutant mice. Some CAII-positive astrocytes and oligodendrocytes developed in the brains of the host CAII-deficient mice at 8, 14 and 18 days posttransplant (DPT). In transplants of either mixed glial cells or oligodendrocyte precursors, CAII-positive oligodendrocytes were less plentiful than CAII-positive astrocytes and appeared to be less healthy. CAII-positive astrocytes developed by 8 DPT, and there were some oligodendrocytes in cerebral cortex at 14 DPT and in brainstem by 18 DPT. The data suggested that if glial-cell progenitors were to be injected into demyelinated lesions, any oligodendrocytes descended from the donor would be accompanied by astrocytes also descended from donor cells.

Animals↗

PubMed1998

Differential uptake of dextran beads by astrocytes, macrophages and oligodendrocytes in mixed glial-cell cultures from brains of neonatal rats.

The present study addresses a controversy over the abilities of astrocytes to perform phagocytosis. Primary glial-cell cultures were prepared from the brains of neonatal rats and were incubated with fluorescently-labeled dextran beads (molecular weights approximately 10 and approximately 40 kDa). Astrocytes and oligodendrocytes were double-labeled by immunofluorescence staining of cell-specific markers, and microglia by lectin histochemistry. Cells were permitted to take up beads for 1 h, fixed, and incubated with primary antibodies, followed by fluorescent secondary antibodies or fluorescently-labeled lectin. Macrophages and astrocytes internalized beads of both sizes. In astrocyte processes the beads appeared to line up along glial filaments. The results, which provide direct evidence for uptake of beads by astrocytes in vitro and against equally rapid, if any, uptake by oligodendrocytes, bear upon issues of acid/base balance and glial cell development and are relevant to neuropathological observations in human disease.

Animals↗

PubMed1998

Glial-cell cultures from brains of carbonic anhydrase II-deficient mutant mice: delay in oligodendrocyte maturation.

Carbonic anhydrase II (CAII) is a multifunctional enzyme found in oligodendrocytes and astrocytes in normal mouse brains. We have begun to compare the glial cells in primary cultures from neonatal genetically CAII-deficient (Car) mice to those from normal (con) mice in order to detect developmental defects, if any, in Car glial cells. In con cultures intensely CAII-positive cells costained with antibodies against the oligodendrocytic markers, O4 and myelin basic protein (MBP), respectively. Most (82%) of the CAII-positive cells were O4-positive, but only approximately 60% were MBP-positive. Some clumps of GFAP-positive cells were CAII-positive. At each respective number of days in vitro (DIV) total numbers of O4-positive cells were similar in Car and con cultures, and total numbers of galactocerebroside-positive cells also were similar in Car and con cultures. However, compared to cells in con cultures at 7 DIV, a lower percent of Car cells in the oligodendrocyte lineage expressed MBP, and morphological differentiation also was subnormal in that the Car cells showed fewer processes and membrane sheets. Car and con cultures expressed similar numbers of MBP-positive cells by 10 DIV. The results suggest a temporary delay in the maturation of Car oligodendrocytes.

Animals↗

PubMed1997

Rapid upregulation of the Pi isoform of glutathione-S-transferase in mouse brains after withdrawal of the neurotoxicant, cuprizone.

Cuprizone intoxication has been used as a model for reversible demyelination in the CNS. During the course of cuprizone intoxication, the glutathione-S-transferase isoform, Pi, normally and oligodendrocytic marker, appears in reactive astrocytes (Cammer ad Zhang, 1993). The present experiments address the changes in expression of Pi after removal of cuprizone from the diet of the affected mice. In order to localize Pi message, a riboprobe was prepared and in situ hybridization (ISH) performed. Western blots and immunocytochemistry were used to examine Pi protein and other glial cell markers. The data indicated that Pi protein increased during the first 2 d after withdrawal of the toxicant, when the level of the myelin marker, 2',3'-cyclic nucleotide-3'phosphohydrolase, remained minimal. Results of ISH suggested that levels of Pi message in the corpus striatum decreased during cuprizone feeding and began to recover within 2d after withdrawal of the toxicant. Both microglia and astrocytes appeared during the first week of cuprizone administration and persisted during two to three additional weeks on cuprizone. Reactive astrocytes remained in the tissue for at least 6 wk after cuprizone was withdrawn, while microglia receded within days. The findings suggest that astrocytes continue to express Pi after withdrawal of cuprizone.

Animals↗

PubMed1997

Expression of carbonic anhydrase IV in carbonic anhydrase II-deficient mice.

Chronic metabolic acidosis (CMA) in the rabbit upregulates carbonic anhydrase (CA) IV in the proximal convoluted tubule (PCT). This study was designed to assess CA IV expression in a model of CMA in the mouse, i.e., congenital deficiency in CA II [CA(II)D]. In female CA(II)D mice, CA IV specific activity but not CA IV immunoreactivity was upregulated in the renal cortex, specifically in microdissected PCTs. Western blot analysis showed higher expression of CA IV immunoreactive protein in renal membranes from males than in those from females.

Animals↗

PubMed1996

Ganglioside GD3 in radial glia and astrocytes in situ in brains of young and adult mice.

Ganglioside GD3 occurs in immature cells in the neuroectoderm. However, with regard to particular cellular locations of GD3, rat brain has received more attention than mouse brain. In brains from neonatal mice the most intense GD3 immunostaining appears to occur in structures that differ from those that immunostain the most intensely in brains from neonatal rats (Cammer and Zhang: J Histochem Cytochem 44: 143-149, 1996). In the present study epifluorescence and confocal microscopy were used for the purpose of identifying the types of GD3-immunopositive structures in brains of neonatal, 2-week-old, and adult mice. Vibratome sections from mouse brains were double immunostained for GD3 and respective markers for macrophages, microglia, and cells belonging to the oligodendrocyte lineage. Surprisingly, none of those marker antigens immunostained intensely in the same respective structures as GD3. The GD3-positive structures, however, did resemble protoplasmic astrocytes and radial glia, some with GD3-positive end-feet at the glia limitans; however, we did not rule out the possibility that there might be some GD3 on the surfaces of prooligodendroblasts. The scarcity of glial fibrillary acidic protein (GFAP)-positive cells in brains of neonatal mice made it impractical to look for GD3+/GFAP+ structures that might belong to the astrocyte lineage. The Mu subunit of glutathione-S-transferase (Mu) was shown to label radial glia and the few GFAP-positive cells in brains of neonatal mice. Subsequently, confocal microscopy showed Mu and GD3 to be colocalized in radial glia and protoplasmic astrocytes in the neonate. In brains from mice > or = 2 weeks of age GD3 immunostaining was demonstrated in GFAP-positive astrocytes, including reactive astrocytes. Much of the GD3 appeared to occur at the tips of astrocyte processes. It is suggested that GD3 in radial glia and astrocytes may function as a ligand enabling recognition of those structures by neurons or as a precursor of more complex gangliosides in neurons.

Aging↗

PubMed1996

Expression of carbonic anhydrase II mRNA and protein in oligodendrocytes during toxic demyelination in the young adult mouse.

The aim of this study was to identify events that might take place in oligodendrocytes early in the process of demyelination, i.e., before the occurrence of massive loss of myelin. It was considered important to focus on demyelination and remyelination in young adults, in whose brains there would be relatively few juvenile glial precursor cells. CAII mRNA and protein were used to monitor changes in oligodendrocytes during cuprizone intoxication in the mice. After four or eight weeks of cuprizone feeding CAII message became less plentiful in oligodendrocyte processes. Two days after removal of cuprizone CAII message had appeared in those cell processes. Four or eight weeks after beginning cuprizone feeding CAII protein had decreased approximately 25% in forebrain homogenates. The loss of CAII protein was reversible after four weeks on cuprizone, but not after eight weeks. After four weeks of cuprizone feeding the numbers of CAII mRNA-positive oligodendrocytes had decreased by approximately 50%, and after eight weeks, by approximately 80%. By 12 weeks, however, the number of oligodendrocytes expressing CAII mRNA had spontaneously returned to normal levels. Before eight weeks of cuprizone feeding, loss of myelinated tracts in the corpus striatum was reversible. Demyelination appeared to become irreversible after nine weeks of intoxication, although expression of CAII mRNA remained reversible. The results suggest that in the brain of the young adult, oligodendrocytes expressing message for CAII can be generated spontaneously shortly before demyelination becomes irreversible, and can survive and continue to express CAII mRNA but not CAII protein.

Animals↗

PubMed1996

Carbonic anhydrase II in microglia in forebrains of neonatal rats.

In the brains of adult rodents carbonic anhydrase II (CA) immunoreactivity has been observed in the choroid plexus and in oligodendrocytes, astrocytes, and myelin. Localization and functions of CA in the neonatal brain, however, have been controversial. One issue is whether the CAII-immunopositive round and ameboid cells in the corpus callosum and cingulum in the rat CNS during the first postnatal week are oligodendrocytes or microglia. Colocalization of CAII with the microglial antigen, ED1, and the microglia-specific isolectin, BSI-B4, suggested that most (approx. 60%) of the CAII-positive round and ameboid cells in rat brain during the first postnatal week were, indeed, macrophages and microglia. During that initial week, some CAII-positive protoplasmic astrocytes (approx. 40%) were observed as well. At the end of the first postnatal week smooth-surfaced CAII-positive cells began to appear in the corpus callosum. Those cells also bound MAbO4, a marker for the oligodendrocyte cell line. We conclude that during the first postnatal week most of the CAII-positive cells are macrophages and microglia, and that some are protoplasmic astrocytes. During the second postnatal week CAII-positive cells in the oligodendrocyte lineage become apparent, and by the end of that week there are few CAII-positive microglia. Confocal microscopy suggests that in brains of three-day-old rats the ameboid microglia are associated with nerve fibers, where they may perform phagocytosis of axons, directional guidance of axons, or disinhibition of axonal growth.

Animals↗

PubMed1996

A reexamination of ganglioside GD3-immunopositive glial cells in the forebrains of rats and mice less than 1 week of age.

Before examining remyelination in the brains of rats and mice in vivo, we evaluated the criteria for immature cells in both species that belong to the oligodendrocyte lineage. In the forebrains and cerebella of neonatal rats, ganglioside GD3 has been used as an early immunocytochemical marker for such cells. Using tissue sections from brains of perinatal rats and neonatal mice, we performed double immunofluorescence staining of GD3 with markers for brain macrophages and microglia (both termed "microglia" below) and for the early oligodendrocyte marker O4, respectively. In the rat brains, intense GD3 immunofluorescence was observed in cells also bearing the microglial marker ED1, but not in O4-bearing immature oligodendrocytes. In the rat corpus callosum there also were faintly GD3-positive longitudinal processes that did not co-stain with ED1 or BSI-B4. Some additional GD3-positive structures were observed, mostly in gray matter. In the brains of neonatal and 3-day-old mice, the most intensely GD3-positive structures were elongated longitudinal and radial processes, and the BSI-B4-positive microglia were GD3-negative. In both rat and mouse brains there were at least two additional types of GD3-positive structures, which were ovoid or had a diaphanous appearance, respectively. The present results suggest that there are GD3-positive microglia in immature rat brains and, in both rat and mouse brains, GD3-positive structures that have not yet been identified precisely but which may be radial glial or microglial processes. Accordingly, it is inappropriate to use GD3 as a specifically oligodendrocytic marker in vivo.

Animals↗

PubMed1995

Effects of carbonic anhydrase II (CAII) deficiency on CNS structure and function in the myelin-deficient CAII-deficient double mutant mouse.

In the choroid plexus carbonic anhydrase II (CAII) supports the transport of bicarbonate ions, sodium ions, and water from blood to the CSF, and in the myelin sheath CAII supports compaction of myelin by stimulating cotransport of ions and water out from between the myelin membranes. In view of the latter, it is surprising that mutant mice deficient in CAII (Car-2n) have compact myelin. Since myelin basic protein also takes part in myelin compaction, we bred double CAII-deficient, myelin-deficient (Mld) mutant mice, in which the adults would have some compact myelin sheaths and a partial deficiency in myelin basic protein, with a view to examining oligodendrocytes and myelin sheaths in the double mutant. Like the parent Mld strain, the double mutants displayed tremors and seizures; however, the onset of seizures was delayed significantly in the double mutants, and the lifespan increased by several months. Like the brains of Car-2n mutants, those of double mutants (MldCar-2n) were deficient in mRNA and protein for CAII and showed upregulation of a different isozyme, CAIV. In the double mutants, oligodendrocytes were reduced in number, and the myelin sheaths and oligodendrocytes were swollen. The partial protection against seizures, which CAII deficiency conferred, suggests that acidosis in the central nervous system (CNS) of the Car-2n and MldCar-2n mice, due to absence of CAII from the choroid plexus, may downregulate the activity of NMDA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Up-regulation of carbonic anhydrase isozyme IV in CNS myelin of mice genetically deficient in carbonic anhydrase II.

Carbonic anhydrase (CA) II is the major CA isozyme in the brain, where it participates in acid-base homeostasis, fluid transport, and myelin synthesis. The CA II deficiency [CA(II)D] mutation in the mouse results in structural changes in the glial cells in the CNS and in decreased susceptibility to seizures, but no detectable changes in myelin yield and ultrastructure. We compared the CA isozymes in brain and spinal cord fractions, as well as in purified myelin, between CA(II)D and control mice. CA(II)D resulted in a much lower total CA specific activity in all tissues examined but in higher CA IV specific activities in soluble and membrane-associated fractions and pure myelin. Western blots of purified myelin showed a band corresponding to CA IV in CA(II)D mice. This band was weak or undetectable in myelin samples from normal mice. Immunocytochemical staining demonstrated CA IV in oligodendrocytes and myelinated tracts in normal mouse brains and stronger staining of the same structures in brains of CA(II)D mutants. We conclude that CA(II)D mutation in the mouse up-regulates CNS CA IV. We speculate that this up-regulation could mitigate the effect of CA(II)D on myelin formation and maintenance.

Animals↗