The effects of thyroid hormone on neural development in vitro were studied using rat fetal forebrain aggregating cultures. They were examined morphologically after growth for 21 days in medium containing fetal calf serum (S+), in a chemically defined medium (S-), or in serum-free medium containing 30 nM triiodothyronine (T3). Aggregates grown in S+ showed certain morphological differences compared to those grown in the absence of serum: a glia limitans was present in the former, but not the latter, which were further characterized by a marginal zone rich in fibres and containing few cells. Immunocytochemistry using a monoclonal antibody against neurofilaments showed that immunostaining was most pronounced in aggregates grown in T3 (especially in the marginal zone) and weakest in those grown in S+. Quantitative estimation using an immunoadsorbent assay confirmed that T3 medium increased the amount of neurofilament protein in the aggregates, consistent with the view that thyroid hormone promotes neural development in vitro.

Animals↗

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In vitro preclinical lead optimisation technologies (PLOTs) in pharmaceutical development.

The explosion of genuine high throughput technologies has allowed large compound libraries to be screened with ever-increasing biological specificity, exacerbating the problem of lead candidate selection for subsequent drug development. To avoid creating a bottleneck, compounds identified from the high throughput screens undergo lead optimisation by employing medium-throughput screen which permits ranking in terms of their basic absorption, distribution, metabolism, excretion (ADME) and toxicological properties. The historical role of the CRO in the drug discovery/development continuum has been to perform efficacy and toxicology studies, simply to support the regulatory submission of lead candidates. This situation is, however, changing with the development of preclinical lead optimisation technologies facilitating the selection of leading candidates, thereby bridging the gap between high throughput efficacy screens and conventional safety assessment programmes.

Drug Approval↗

PubMedDate not supplied

Brain reaggregate cultures in neurotoxicological investigations: adaptational and neuroregenerative processes following lesions.

In vitro neural systems can be predictive for CNS neurotoxicity, except where xenobiotics primarily affect the blood-brain barrier. The wide range of systems now used in neurobiological studies is available for mechanistic neurotoxicological investigations although the choice of system is generally arbitrary. A more rational approach may now be justified. There are many culture systems available including neural cell lines, organotypic explant or reaggregation cultures, and primary monolayer cultures of individual neural cell types: neurons, astrocytes, and oligodendrocytes. Of these models much success has recently been achieved using the organotypic explant culture type. Similarly in our laboratories, using rat whole-brain reaggregate cultures, we have demonstrated good in vitro/in vivo correlations for the cholinergic neurotoxicant ethylcholine mustard aziridinium (ECMA) where specific cholinergic lesions are produced using low concentrations of ECMA (12.5 microM). Higher concentrations (25-50 microM) were more cytotoxic, as shown, for example, by nonspecific effects on cerebellar glutamatergic granule neurons. Treatment of reaggregates lesioned with the cholinotoxin with a neurotrophic factor, nerve growth factor (NGF), did not reverse the lesion but treatment of control cells with NGF (50 ng/ml) elevated both choline acetyltransferase (ChAT) activity and muscarinic receptor binding. The "lesioned" reaggregate culture system may thus be of future value in evaluating potential therapeutic agents that could reverse such lesions in the CNS. By supplementing the information gained in the reaggregate system with tests using primary monolayer cultures of neurons or astrocytes we can propose a stepwise screening system for potential neurotoxicants in vitro. In its simplest form this is (1) screen initially using tumor-derived neural cell line, (2) test selected compounds in whole-brain reaggregates, and (3) supplement information with primary monolayer cultures of individual neural cell types.

Adaptation, Physiological↗

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Gliotoxicity in brain reaggregate cultures caused by oxidants and excitatory amino acids can be prevented by alpha-tocopherol and MK-801.

Glutamine synthetase (GS) is a key enzyme involved in glutamate compartmentalisation which may be pivotal in the course of both central free-radical mediated and excitotoxic events. The ability of the oxidants FeCl2 and H2O2 and the excitatory amino acid, N-methyl-D-aspartate (NMDA) to induce changes in astrocytic GS and glial fibrillary acidic protein (GFAP), were assessed in whole rat brain reaggregate cultures. Both FeCl2 and H2O2 reduced GS activity whereas NMDA produced a large increase in enzyme activity. GFAP was not altered significantly by either oxidant although NMDA increased the level of this protein. These effects on such astroglial markers could be reversed in vitro following exposure to a-tocopherol (FeCl2 and H2O2) and MK-801. This study therefore demonstrates that inactivation of GS can be caused by free radical insult whereas stimulation of brain GS and reactive gliosis is produced by excitatory amino acids acting at neuronal NMDA receptors. The study of these gliotoxic events in 3-dimensional reaggregate cultures suggests that this model may be used to detect neuroprotective effects of novel pharmacological agents.

Animals↗

PubMedDate not supplied

Rat brain mast cells: an in vitro paradigm for assessing the toxic effects of neurotropic therapeutics.

Neurotrophic factors (NTFs) such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) are currently being explored as novel therapeutics in a range of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. To this end, animal studies and clinical trials have been conducted to assess the toxic effects of recombinant NTFs. It is apparent that both NGF and BDNF induce a range of adverse effects, for example inflammation, hyperalgesia, and disturbances in CNS biogenic amine levels which variously manifest as weight loss/gain, changes in feeding behaviour and general malaise. It has been demonstrated that NGF induces release of biologically active mediators, such as histamine, from rat peritoneal mast cells (RPMC). However, whether other NTFs do likewise or indeed are able to induce secretion from other mast cells types had not been explored. We have developed a novel protocol for dispersing mast cells from rat brain tissue, in particular the thalamus which contains the highest number of mast cells in the adult rat. Rat brain mast cells (RBMC) released histamine in a concentration dependent manner in response to NTFs, with a rank order of BDNF > CNTF > NGF; in contrast RPMC were refractory to the effects of BDNF and CNTF. The ability of NTFs to induce release of histamine (a neurotransmitter and neuromodulator in the CNS) from RBMC may go some way to explain some of the adverse effects apparent in vivo upon dosing with NTFs. Mast cells in vitro, and brain mast cells in particular, offer the potential to screen novel NTFs for their neuroimmunotoxic potential relevant to detecting likely clinical adverse effects in humans.

Animals↗