Neurotoxicologyand Teratology, Vol. 13, pp. 401-406. ©Pergamon Press plc, 1991. Printed in the U.S.A. 0892-0362/91 $3.00 + .00 Total Number and Mean Cell Volume of Neocortical Neurons in Rats Exposed to 2,5-Hexanedione With and Without Acetone POUL STRANGE,* ARNE MOLLER,*t I OLE LADEFOGED,:~ HENRIK R. LAM,$ JENS-JORGEN LARSEN$ AND PETER ARLIEN-SOBORG* Received 2 July 1990 *Neurological Research Laboratory, Hvidovre University Hospital, Copenhagen "PStereological Research Laboratory, Aarhus University ~:Institute of Toxicology, National Food Agency, SOborg, Denmark STRANGE, P., A. MOLLER, O. LADEFOGED, H. R. LAM, J.-J. LARSEN AND P. ARLIEN-SOBORG. Total number and mean cell volume of neocortical neurons in rats exposed to 2,5-hexanedione with and without acetone. NEUROTOXICOL TERA- TOL 13(4) 401-406, 1991.--The toxicological effects of 2,5-hexanedione (2,5-HD) alone and combined with acetone on the number and size of neurons in the cerebral cortex of rats were evaluated with stereological techniques. Thirty rats were equally divided into three groups: One control, one receiving 0.5% 2,5-HD, and one receiving 0.5% 2,5-HD and 0.5% acetone in the drinking water for seven weeks. Unbiased estimates of the total number of neocortical neurons, as well as the mean neuronal nuclei and cell body volumes were obtained from systematically sampled 3.5-1xm sections. The total number of neurons in the 2,5-HD group was significantly smaller than the control group (p<0.05, one-tailed t-test). Both test groups showed significant changes in the mean cell body volume: Compared with the control group, animals exposed to 2,5-HD had 11% smaller cell body volumes while animals exposed to 2,5-HD and acetone had 13% larger cell body volumes. These data represent the first unbiased estimation of mean cell volume in toxicology. We propose the nucleator method as an efficient and accurate tool for estimating quantitative changes in toxicological research. Organic solvents 2,5-Hexanedione Acetone Rat Neurons Total neuron number Neuron size Stereology Disector Nucleator Unbiased THE oxidized product of methyl-butyl-ketone (MBK) and n-hex- ane is 2,5-hexanedione (2,5-HD), a known neurotoxin (1,13). Previous studies have focused primarily on the morphological and functional effects of 2,5-HD in the peripheral nervous sys- tem (PNS) (23) although morphological changes have been re- ported in the central nervous system (CNS) (22). Few studies have focused on neuronal changes in the CNS. These suggest a remodelling effect, perineuronal cell reaction and neuronal death following exposure to 2,5-HD (25,27). The most striking mor- phological effect ascribed to n-hexane, MBK, and their common metabolite 2,5-HD, is multifocal paranodal axonal swellings filled with neurofilaments in the terminals of larger axons. Para- nodal myelin-thinning and retraction is often found whereas qualitative descriptions of changes in the cell body (25,27) have shown few, if any, obvious alterations (3, 5, 21). Potentiation of the neurotoxic effect of n-hexane and 2,5-HD MEK has been demonstrated clinically and in experimental ani- mals (2,20). In the PNS the toxic effect of 2,5-HD in rats is potentiated by acetone (15). The purpose of the present study in the CNS is to investigate the toxicity of 2,5-HD, alone or in combination with acetone. The complete study includes stereo- logical, biochemical and behavioral parameters. In this paper emphasis is placed on the stereological analysis, while biochem- ical and behavioral parameters are reported by Lain et al. (16). Until recently there have been two major problems in reli- ably estimating parameters in 3 dimensions using 2-dimensional probes, the most serious of which has been the necessity to make assumptions about shape and size of particles which do not re- flect the known true condition, thus biasing the results. The other major problem has been lack of quantitation efficiency, leading to excessive data collection. Both problems have now been solved by a relatively new multidisciplinary science called stereology (9,10). For the sake of simplicity, we only describe methods for the unbiased estimation of neuron number and mean volume in the rat neocortex. The methods are applicable, however, to an infi- nite number of other settings, ranging from quantitative analysis of crystals in a piece of rock, to the influence of mean cell wall thickness and cell size on the total strength of a specific plant fibre. These stereological methods have made possible estimates of the total number of cells in microscopic structures, as well as unbiased estimates of mean cell volume. Changes of this type are of particular interest in neurotoxicology, as neurons cannot- duplicate in a mature brain, and since cell alternations precede ~Requests for reprints should be addressed to Ame MOiler, Neurological Research Laboratory, Barholin Instituttet, Kommune Hospitalet, DK-1399, Copenhagen, Denmark. 401