Cell and Animal Models in Aging and Dementia Research

268 Pages·1994·7.047 MB·English

by  H. Oberpichler-Schwenk (auth.), Prof. Dr. S. Hoyer, Dr. D. Müller, Dr. K. Plaschke (eds.)

loumalof
Neural
Transmission
Supplement 44
S. Hoyer, D. Muller, and K. Plaschke (eds.)
Cell and Animal Models
in Aging and
Dementia Research
Springer-Verlag Wien New York
Prof. Dr. S. Hoyer
Dr. D. M tiller
Dr. K. Plaschke
Institute for Pathochemistry and General Neurochemistry,
University of Heidelberg, Heidelberg, Federal Republic of Germany
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© 1994 Springer-Verlag / Wien
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Printed on acid-free and chlorine-free bleached paper
With 63 Figures
ISSN 0303-6995
lSBN-13: 978-3-211-82549-5 e-lSBN-13: 978-3-7091-9350-1
DOl: 10.1007/978-3-7091-9350-1
Preface
The increasing life expectancy of the population in nearly all highly industrialized
countries is not accompanied with physical and/or mental health in any case.
With respect to mental function, this is increasingly compromised by abnormal,
age-related brain disorders causing a loss in intellectual capacities, i.e., in learn
ing and memory. This dementia state forms one of the greatest burdens in
medical and socio-economic terms because most of the early pathogenetic fac
tors of the age-related dementias are still unknown, and, therefore, a successful
therapy is not possible at present.
The contribute to the understanding of genetic, molecular, cellular, mor
phological and behavioral aspects of normal aging and age-related disorders,
a workshop "Cell and Animal Models in Aging and Dementia Research" was
held in Heidelberg, Federal Republic of Germany, on September 2nd and 3rd,
1993. The workshop was organized in the frame of the IPA Congress in Berlin,
and was generously supported by the Hirnliga e. V. (Brain League). The data
presented at the workshop deriving from cell and animal models covered many
actual aspects of both normal and abnormal brain aging, and its interactions.
It is the hope of the authors that the results from these models can help to fill
the gap which exists in the understanding of early events of mental aging and
age-related (dementing) disorders.
S. HOYER, Heidelberg
Contents
Oberpichler-Schwenk, H., Krieglstein, J.: Primary cultures of neurons for testing neu-
roprotective drug effects ............................................... .
Nitsch, R. M., Slack, B. E., Farber, S. A., Schulz, J. G., Deng, M., Kim, C., Borghesani,
P. R., Korver, W., Wurtman, R. J., Growdon, J. H.: Regulation of proteolytic
processing of the amyloid ~-protein precursor in Alzheimer's disease in transfected
cell lines and in brain slices .............................................. 21
Smith-Swintosky, V. L., Mattson, M. P.: Glutamate, beta-amyloid precursor proteins,
and calcium mediated neurofibrillary degeneration .......................... 29
Chatterjee, S. S., Noldner, M.: An aggregate brain cell culture model for studying
neuronal degeneration and regeneration ................................... 47
Diekmann, S., Nitsch, R., Ohm, T. G.: The organotypic entorhinal-hippocampal com
plex slice culture of adolescent rats. A model to study transcellular changes in a
circuit particularly vulnerable in neurodegenerative disorders ................. 61
Schubert, P., Keller, F., Nakamura, Y., Rudolphi, K.: The use of ion-sensitive electrodes
and fluorescence imaging in hippocampal slices for studying pathological changes
of intracellular Ca2 + regulation .......................................... 73
Blass, J. P.: The cultured fibroblast model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 87
Bubna-Littitz, H., Jahn, J.: Psychometric testing in rats during normal ageing. Pro-
cedures and results ..................................................... 97
Struys-Ponsar, C., Florence, A., Gauthier, A., Crichton, R. R., van den Bosch de Aguilar,
Ph.: Ultrastructural changes in brain parenchyma during normal aging and in
animal models of aging ................................................. III
Kadar, T., Arbel, I., Silbermann, M., Levy, A.: Morphological hippocampal changes
during normal aging and their relation to cognitive deterioration ............. 133
Muller, W. E., Stoll, S., Scheuer, K., Meichelbock, A.: The function of the NMDA-
receptor during normal brain aging ....................................... 145
Benzi, G., Gorini, A., Arnaboldi, R., Ghigini, B., Villa, R. F.: Age-related alterations
by chronic intermittent hypoxia on cerebral synaptosomal ATPase activities .... 159
Arendt, T.: Impairment in memory function and neurodegenerative changes in the
cholinergic basal forebrain system induced by chronic intake of ethanol . . . . . . .. 173
Pepeu, G., Marconcini Pepeu, I.: Dysfunction of the brain cholinergic system during
aging and after lesions of the nucleus basalis of Meynert .................... 189
Schliebs, R., Feist, T., Rofiner, S., Bigl, V.: Receptor function in cortical rat brain
regions after lesion of nucleus basalis ..................................... 195
Hellweg, R.: Trophic factors during normal brain aging and after functional damage 209
Czech, Ch., Masters, c., Beyreuther, K.: Alzheimer's disease and transgenic mice 219
Chessell, I. P., Francis, P. T., Webster, M.-T., Procter, A. W., Heath, P. R., Pearson,
R. C. A., Bowen, D. M.: An aspect of Alzheimer neuropathology after suicide
transport damage ...................................................... 231
VIII Contents
Hortnagl, H.: AF64A-induced brain damage and its relation to dementia ......... 245
Hoyer, S., Milller, D., Plaschke, K.: Desensitization of brain insulin receptor. Effect
on glucose/energy and related metabolism ................................. 259
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Listed in Current Contents
J Neural Transm (1994) [Suppl] 44: 1-20
© Springer-Verlag 1994
Primary cultures of neurons for testing neuroprotective
drug effects *
H. Oberpichler-Schwenk and J. Krieglstein
Institut fUr Pharmakologie und Toxikologie, Philipps-Universitat, Marburg,
Federal Republic of Germany
Summary. Primary cultures of neurons are widely used for the investigation
of pathomechanisms of neuronal damage und for the evaluation of neuro
protective drug effects. The present paper gives a short survey of frequently
used primary neuronal culture systems and of experimental measures for the
induction of defined neuronal damage with particular respect to the patho
mechanisms of cerebral ischemia. Neuroprotective durg effects as achieved
under these conditions are reviewed, and the neuroprotective effects of
glutamate antagonists, radical scavengers, and neural growth factors are
discussed in some more detail.
Introduction
Neurodegeneration is a common feature of disorders such as stroke, multi
infarct dementia, Alzheimer'S, Parkinson's and Huntington's disease. Since
the number of people affected by these disorders is steadily increasing, there is
a great demand for a rational drug therapy. To this end, clarification of the
pathophysiological events leading to neurodegeneration is of utmost import
ance, not only for the understanding of the pathogenesis but also as a
prerequisite for the specific development of neuroprotective drugs.
Primary cultures of neurons provide important tools for this task.
Neuronal reactions to specific changes of the extracellular milieu can be
investigated on the cellular level. Thus, neuronal reactions can be differen
tiated from non-neuronal reactions. Owing to the absence of the blood
brain barrier, drug effects can be tested without pharmacokinetic considera
tions. A further general advantage of cultured cells may be seen in the
reduction of the number of animal experiments. In this context, primary
neuronal cultures, although they still require a certain number of donor
* Abbreviations: AMPA a-Amino-3-hydroxy-5-methylisoxazole-4-propionate; APV
2-Amino-5-phosphonovaleric acid; eNQ X 6-Cyano-7 -nitroquinoxaline-2 ,3-dione;
DNQX 6,7-Dinitroquinoxaline-2,3-dione; LDH Lactate dehydrogenase; NBQX 2,3-
Dihydro-6-nitro-7 -sulphamoyl-benzo( f)quinoxaline; N M DA N -Methyl-D-aspartate
2 H. Oberpichler-Schwenk and J. Krieglstein
animals, are nevertheless often preferred to neuronal cell lines. While
neuronal cell lines contain metabolically deviated cells, primary cultures
pertain many morphological and functional features of their in vivo counter
parts as will still be discussed.
In the following we shall discuss some primary neuronal culture models
commonly used for the investigation of neuroprotective drugs. It will be
shown that drug effects evaluated under these in vitro conditions can well
be extrapolated to in vivo conditions, and a few more recent concepts of
neuroprotection in vitro will be presented.
Neuronal cultures in common use
Like for animal experiments, mice and rats are most frequently used as
donor animals for primary neuronal cultures. Cells are derived from nearly
all parts of the central nervous system, such as whole cortex (Dichter,
1978; Choi et al., 1987; Prehn et al., 1993a), visual cortex (Huettner and
Baughman, 1986), hippocampus (Banker and Cowan, 1977; Rothman,
1984; Yamada et al., 1989; Prehn et al., 1993b), striatum (Weiss et al.,
1986), hypothalamus (Yamashita et al., 1992), cerebellum (Messer, 1977;
Balazs et al., 1988; Novelli et al., 1988), and dorsal root ganglia (Scott,
1982). Whereas in most cases the tissue is dissociated into single cells and
the cell suspension is seeded onto a prepared surface, a few authors used
explants of brain tissue and performed experiments on the manolayer of
cells spreading out of the explant (Gahwiler, 1981; Mattson et al., 1988).
Many investigators use nervous tissue from fetal rats or mice for the
preparation of their cultures. In this stage of development, differentiation of
the neurons is still comparatively poor and the cells presumably withstand
the dissociation procedures better than at later stages. Nevertheless, several
authors have successfully utilized postnatal donor animals for the preparation
of neuronal cultures (Messer, 1977; Huettner and Baughman, 1986; Trussell
et al., 1986; Yamada et al., 1989; Zorumski et al., 1989; Prehn et al.,
1993a,b). Even 19-day-old rats yielded good cultures of hippocampal
neurons (Nakajima et al., 1986).
Neurons in dispersed cell culture start to extend several neurites within
one day after plating. These neurites differentiate into an axon and several
dendrites (for references see Sargent, 1989) and, depending on the seeding
density, can form an extensive network. Synaptic contacts have been shown
by electronmicroscopy (Weiss et al., 1986; Harris and Rosenberg, 1993;
Ichikawa et al., 1993) and electrophysiology (Dichter, 1978; Rothman and
Samaie, 1985). With the formation of neurites, the somata of the neurons
take on pyramidal, bipolar or stellate shapes reminiscent of their in situ
counterparts. When seeded at high density (i.e. 100,000 to 300,000 cells/cm2),
the neurites often form aggregates extending many neurites. Apart from
these morphological characteristics, neurons in culture have been shown to
contain neuron-specific markers such as neuron-specific enolase (Soderback
Primary cultures of neurons for testing neuroprotective drug effects 3
et aI., 1989) and microtubule-associated protein 2 (Mattson et aI., 1989) as
well as transmitters like glutamate and gamma-aminobutyric acid (Mattson
and Kater, 1989).
Besides the neurons, cultures from fetal (15 to 20 days of gestation) or
postnatal rat or mouse brain always contain a number of non-neuronal cells,
predominantly astrocytes. Since these non-neuronal cells are still mitotic and
thus may outgrow the neurons, their proliferation is often halted by addition
of an antimitotic agent like cytosine arabinoside or fluorodesoxyuridine
after they have become confluent. Hence, these primary neuronal cultures
are actually mixed neuronal/glial cultures. A certain proportion of glial cells
seems to be necessary for long-term survival of the neurons (Banker and
Cowan, 1977; Banker, 1980), perhaps owing to the release of (a) neuro
trophic factor(s) (Muller and Seifert, 1982). Some authors have seeded cell
suspensions from postnatal nervous tissue (cortex, hippocampus) onto a
prepared glia feeder layer (Huettner and Baughman, 1986; Nakajima et aI.,
1986; Yamada et aI., 1989), although postnatal rat cortical and hippocampal
neurons could also be cultured successfully without such precautions (Furuya
et aI., 1989; Jahr and Stevens, 1987; Prehn et aI., 1993a,b).
Mixed cultures have been used for numerous neurobiological, physio
logical and pharmacological investigations and lend themselves to morpho
logical or electrophysiological evaluations. However, in analyses utilizing
the whole cell mass, as for example measurement of energy metabolites or
protein content, mixed cultures may yield equivocal results, since the non
neuronal cells may react differently to the experimental procedure than the
neurons. Thus, for such biochemical measurements pure neuronal cultures
are preferable. We have used neuronal cultures from chick embryo telen
cephalon for various pharmacologic studies (Krieglstein et aI., 1988;
Ahlemeyer and Krieglstein, 1989; Oberpichler et aI., 1990; Peruche et aI.,
1990; Seif el Nasr et aI., 1990; Prehn et aI., 1992, 1993b). These neuronal
cultures have been first prepared and characterized by Pettmann et ai.
(1979) and Louis et ai. (1981). They are prepared from 7-day-old embryos,
i.e. at a developmental stage before gliogenesis has occurred. Proliferation
of any glioblasts is hampered by polylysine treatment of the culture flasks
and by thorough dissociation of the tissue into single cells. In this way
nearly pure neuronal cultures are obtained with about 98% of the cells
binding tetanus toxin and less than 0.1 % of the cells corresponding to glia
(Pettmann et aI., 1979). Like in the mixed cultures described above, the
neurons form an extensive network of neurites. Membrane thickenings
suggestive of synaptic junctions were found three to four days after pre
paration of the cultures, and characteristic synapses were shown in 6-day
old cultures (Louis et al., 1981). The same authors have shown that the
majority of neurons in these cultures are dopaminergic, containing tyrosine
hydroxylase and DOPA decarboxylase activity, whereas cholineacetyltrans
ferase and glutamate decarboxylase activity remain rather low. Probably
owing to the lack of glial cells, the life span of the neurons is comparatively
short, and cultures start to disintegrate after eight to nine days in vitro
4 H. Oberpichler-Schwenk and J. Krieglstein
(Louis et al., 1981; own unpublished observations). Our experiments with
cultured chick neurons were performed on cultures up to seven days in
vitro.
Evaluation of neuroprotection in primary cultures
In order to evaluate neuroprotective effects in neuronal cultures, the
cultures have to be experimentally damaged in some way. This damage
should then be alleviated by the potentially neuroprotective measures. Since
our interest lies on the pharmacology of cerebral ischemia, we shall con
centrate upon models of experimental neuronal damage with a certain
relation to the pathophysiology of cerebral ischemia, although there are
also other means to impair cultured neurons, like the intoxication with
methylphenyltetrahydropyridine (MPTP).
Neuroprotection under conditions of impaired oxygen availability
In cerebral ischemia, the brain tissue is deprived of both oxygen and
glucose. Oxygen deprivation in vitro was used to damage primary cultures
of rat hippocampal neurons (Rothman, 1984) or mouse cortical neurons
(Goldberg et al., 1987, 1988). Cultures were kept under nominally oxygen
free conditions for several hours and neuronal damage was assessed either
directly after the hypoxic incubation (Rothman, 1984) or after 20 to 24
hours of normoxic recovery (Goldberg et al., 1987, 1988). This procedure
led to broad neuronal damage, as evidenced microscopically by swelling and
vacuolation of the cell somata, loss of phase brightness, and degeneration of
neurites. Damaged neurons could also be identified by trypan blue staining.
Goldberg et al. (1987) additionally quantified the extent of neuronal damage
by measuring the efflux of the cytosolic enzyme lactate dehydrogenase
(LDH) into the culture medium over 24 hours after the hypoxic challenge.
They found a good correlation between the extent of neuronal loss and the
LDH release and could rule out the possibility that a considerable proportion
of the LDH had been released by the glia present in their cultures. How
ever, measurement of LDH release may yield equivocal results under con
ditions where glia are also damaged, since glial cells contain (and, when
damaged, release) more LDH activity than neurons (Juurlink and Hertz,
1993).
Neuronal damage by oxygen deprivation required at least 6 hours of
nominally oxygen-free conditions. This is due to the facts that 1. in the
presence of glucose neurons can survive on minimal oxygen tensions for a
rather long time, whereas 2. under the experimental conditions used, by
flushing the culture dishes with nitrogen it takes about two hours to lower
the oxygen tension in the culture medium to below 1 mmHg (Goldberg et
al., 1986). In order to obtain a more defined deprivation of aerobic metab
olism, several authors including us used blockers of the respiratory chain