Claus Nissen

ACTIVITY AND ISOENZYME PATTERN OF LACTATE DEHYDROGENASE IN ASTROBLASTS CULTURED FROM BRAINS OF NEWBORN MICE

Lactate dehydrogenase activity and isoenzyme distribution was determined in primary cultures of astroblasts as a function of the culture period. The specific activity increased during this period with a peak value (1.91 ± 0.18μmol x min‐1 x mg‐1 cell protein) after 2 weeks in culture. The isoenzyme pattern changed during 3 weeks in culture towards a higher proportion of the H4 (LDH‐1) isoenzyme which is analogous to the in vivo pattern. Omission of serum with or without dBcAMP (0.5 mM) in the culture medium during the third week of culture further enhanced this prominence of the H4 isoenzyme. The specific activity (1.58 × 0.06 μmol x min‐1 x mg‐1 cell protein) of cultures grown in the presence of 0.5 mM‐dBcAMP and absence of serum was close to the activity in the adult brain.

Monochromatic Pupillometry in Unilateral Glaucoma Discloses no Adaptive Changes Subserved by the ipRGCs

Purpose: To detect signs of a possible adaptive mechanism of the intrinsically photosensitive ganglion cells in unilateral glaucoma. Method: Eleven patients with unilateral glaucoma, classified by automated perimetry (glaucoma: mean deviation <0), were studied by monochromatic pupillometry, employing red (660 nm) or blue (470 nm) light, and by optical coherence tomography of the peripapillary retinal nerve fiber layer. The main outcome measure in pupillometry, the area under the curve (AUC), i.e., the product of pupillary contraction amplitude and time, was determined during and after light exposure in glaucomatous and unafflicted fellow eyes and compared to the AUCs of a healthy, age-matched control group. Results: The AUC to stimulation with blue light was significantly reduced in glaucomatous eyes, both during and after stimulus, compared with that of fellow, unafflicted eyes (p ≤ 0.014). The AUC to red light stimulation was reduced during (p = 0.035), but not after (p ≥ 0.072), exposure in glaucomatous eyes. In the unafflicted fellow eyes, the pupillary response to blue light did not differ from that of healthy controls. Conclusion: The pupillary response to blue light was decreased in the glaucomatous eyes of unilateral glaucoma. No difference was detected between the pupillary light response of the unafflicted fellow eyes and that of a healthy, age-matched control group. Thus no sign of an adaptive mechanism was detected, neither in the glaucomatous nor in the unafflicted fellow eyes, and consequently glaucoma appears to differ from non-arteritic anterior ischemic optic neuropathy.

REGULATION OF OXYGEN CONSUMPTION IN NEUROBLASTOMA CELLS: EFFECTS OF DIFFERENTIATION AND OF POTASSIUM

Abstract— The rate of oxygen consumption has been measured micromanometrically in fresh mouse neuroblastoma cells and in corresponding cells cultivated with and without 20% calf serum known to suppress differentiation. Fresh cells and cells cultivated in the serum depleted medium showed a relatively intense respiration (0.5–1.0 × 10−5μl/h/cell), whereas the proliferating, cultivated cells had a low rate of oxygen uptake (0.2 × 10‐5μl/h/cell), provided they did not differentiate morphologically during the 4 h of measurement in the serum‐free diver medium. Both morphological and metabolic differentiation of such cells occurs rapidly but may be completely prevented by siliconization of the divers.

The effect of pupil size on stimulation of the melanopsin containing retinal ganglion cells, as evaluated by monochromatic pupillometry.

Purpose: To evaluate the influence of the size of the light exposed pupil in one eye on the pupillary light reflex of the other eye. Method: Using a monochromatic pupillometer, the left eye in each of 10 healthy subjects was exposed to 20 s of monochromatic light of luminance 300 cd/m2, first red (660 nm) and in a following session, blue (470 nm) light. The consensual pupillary diameter in the right eye was continuously measured before, during, and after light exposure. Subsequently, Tropicamide 1% or Pilocarpine 2% was instilled into the left eye and when the pupil was either maximally dilated or contracted, the entire sequence of red and blue light exposure repeated. After at least 3 days, when the effect of the eye drop had subsided, the entire experiment was repeated, this time employing the other substance. Results: Prior dilatation of the left pupil augmented the post light contraction to blue (p < 0.0001), but not to red light. The contraction during light exposure did not change. Prior contraction of the left pupil decreased the post-stimulus contraction to blue light (p < 0.04). Conclusion: The size of the light exposed pupil influences the magnitude of the response to blue, but not to red light. Prior dilatation may therefore prove useful, when the response to blue light – as a marker of melanopsin containing retinal ganglion cell function – is of interest, especially when this response is weak.

Dissociation of Pupillary Post-Illumination Responses from Visual Function in Confirmed OPA1 c.983A > G and c.2708_2711delTTAG Autosomal Dominant Optic Atrophy

Purpose: To test whether the melanopsin-containing, intrinsically photosensitive retinal ganglion cells (ipRGCs), as evaluated by examination of the pupillary light reflex (PLR), are preserved in genetically confirmed autosomal dominant optic atrophy (ADOA). Method: Twenty-nine patients with either the c.983A > G (n = 14) or the c.2708_ 2711delTTAG mutation (n = 15) were examined with monochromatic pupillometry, using isoluminant (300 cd/m2), red (660 nm) or blue (470 nm) light, optical coherence tomography, automated visual field analysis, and with determination of best corrected visual acuity (BCVA). Since we examined two different mutations, initially we compared all outcome variables between the two, and finding no statistically significant difference, pooled them. Results: Despite a poor BCVA (56 letters, ETDRS) in the ADOA patients, their post-illuminatory pupil responses did not differ significantly from those of healthy controls (blue, p = 0.45, red, p = 0.49, t-test), and no statistically significant effect was noted of peripapillary retinal nerve fiber layer thickness, ganglion cell-inner plexiform layer thickness, or age. Conclusion: The PLR to blue light of high luminance (300 cd/m2) was preserved in both c.983A > G and c.2708_2711delTTAG ADOA despite severe visual loss and optic nerve atrophy. The study confirms, in a large sample of two genetically homogenous groups, that the ipRGCs are spared in ADOA.

Rate of oxygen uptake by the N N cell line of hamster astroglia: lack of effect by excess potassium

GLIA cell function and metabolism have attracted considerable interest during recent years. Investigations are, however, hampered by difficulties in obtaining intact cells from living animals in sufficient amounts, and cultured cells have been resortcd to. Both primary cultures and established cell lines have been used. An easily replicable, commercially available preparation is the spontaneously transformed astroglia line, N N , which originally was developed from the brains of new-born hamsters (Smm et al., 1970; EICHBERG et a/. , 1971; SILBERSTEIN et al., 1972). These cells have been well characterized with respect to their content of Na’ and K + (LEES & SHEIN, 1970; GILL et al., 1974), of free amino acids (MOKRASCH, 1971), and of nuclcic acids and glycolipids (SHEIN et al., 1970; ROBERT et al., 1975) as well as the uptake kinetics for chloride (GILL e t al., 1974), and the activities of several enzymes including the Na+-K+-ATPase (EMBREE et al., 1971; MANDEL et al., 1974). No information seems, however, to be available about the energy metabolism, and the purpose of the present work has been to study the oxygen uptake of the N N cells. This was done using either whole cultures or microsamples of selected cells and after cultivation in both the absence and the presence of 0 1 mM-dibutyryl-CAMP (BcAMP), which enhanccs the biochemical maturation of primary cultures of glia cells (SCHOUSBOE et a/., 1975). The oxygen consumption was followed during incubation in both ‘physiological’ and potassium-rich media, because the respiration of mature brain-cortex tissue is increased by a substantial elevation of the K + concentration in the medium (ASHFORD & Drxo~, 1935; DICKENS & GREVILLE, 1935; HIMWICH et al., 1942). This response seems to occur in glia cells (HERTZ, 1966; ALEKSIDZE & BLOMSTRAND, 1969; HALJAM~E & HAMBERGER, 1971; HERTZ e t al., 1973).

Prior Light Exposure Enhances the Pupil Response to Subsequent Short Wavelength (Blue) Light

Background and Purpose: The photo pigment melanopsin initiates cell depolarization in response to highintensity, short-wavelength light. Antecedent long-wavelength light may potentiate regeneration of the melanopsin photo pigment, We investigated the influence of red or blue exposure on the pupil response to subsequent blue light. Methods: Nine healthy subjects were examined using chromatic pupillometry. With a sequence of 3 consecutive blue exposures or a sequence in which the middle exposure was red light, both sequences repeated in the darkadapted state. The summed pupil response during light was obtained as the area under the curve and the percentage difference (diff %) between the first and last blue stimulus was calculated for each sequence. Findings: The pupil response to the third blue exposure was greater than to first blue light. No significant difference was seen in the diff% when comparing a sequence with a blue intervening versus red intervening light, in the light adapted (P = 0.39) or dark adapted state (P = 0.58). Conclusion: Prior light exposure enhances the pupil response to subsequent blue light stimulation, no differential effect was found between blue and red light. This study suggests that antecedent light history is important when designing protocols and evaluating results of chromatic pupillometry.

Some Histochemical, Biochemical, and Pharmacological Aspects of Differentiation of Neuroblastoma Cells of Mouse

C1300 neuroblastoma cells in vitro retain the ability of neuronal differentiation (Augusti- Tocco and Sato, 1969; Schubert et al., 1969). This maturation of neoplastic immature neuroblasts can be induced by various molecular environments: suppression of fetal serum in the culture medium (Seeds et al., 1970) incubation with BrdU (Schubert and Jacob, 1970) or BcAMP (Furmanski et al., 1971), X-ray irradiation (Prasad, 1971), cultivation with NGF (Hermetet et al., 1972a). Different cellular events were used as proofs of neuronal differentiation: growth of expansions and the presence of enzymes involved in the biosynthesis of neurotransmitters (Augusti-Tocco and Sato, 1969); changes in oxidative metabolism (Ciesielski-Treska et al., 1972); neuronal type pharmacological reactivity (Hermetet et al., 1972b). The cultures of neuroblastoma cells may be useful for genetic mapping, in studying the maturation of neural morphology and function, as well as the effects of the molecular microenvironment.

Genotype–phenotype heterogeneity of ganglion cell and inner plexiform layer deficit in autosomal-dominant optic atrophy

To describe the thickness of the combined ganglion cell and inner plexiform layers (GC‐IPL) and the peripapillary retinal nerve fibre layer (RNFL) in patients with OPA1 c.983A>G or c.2708_2711delTTAG autosomal‐dominant optic atrophy (ADOA).

Unidirectional influx and phosphorylation of glucose analogues in cultured astroblasts

Unidirectional influx of14C-3OMG (3-O-methyl-d-glucose) and rate of phosphorylation of14C-2DG (2-deoxy-d-glucose) were determined in primary cultures of astroblasts under conditions with neglible unstirred layers. The influx exhibited rate constants between 7.2 and 8.3 min−1 in the concentration range 2.5–25 mM of unlabeled 3OMG and was considered constant, irrespective of concentration of 3OMG. The rate of phosphorylation of14C-2DG declined for rising concentrations of 2DG and hence showed saturability. The rate constants ranged from 7.9 to 0.1 min−1 in the concentration range 0.04–25 mM of 2DG. These results are not consistent with the view that the influx limits the rate of phosphorylation. They support the notion that the influx is not rate limiting for the phosphorylation.