Effects of formaldehyde exposure on granule cell number and volume of dentate gyrus: A histopathological and stereological study

Abstract

The hippocampal formation is a complex region of the brain related to memory and learning. The purpose of the present study was to determine whether exposure of neonatal rats to formaldehyde (FA) had either early or delayed effects on the numbers of granule cells in the dentate gyrus (DG). After birth, the neonatal male Wistar rats were exposed throughout a 30-day period to various concentrations of FA: 0 (control group), 6 ppm (low concentration group) and 12 ppm (high concentration group). This was done by placing them for 6 h/day and 5 days per week in a glass chamber containing FA vapor. Then, five animals from each group were anesthetized and decapitated on postnatal day (PND) 30, and the remaining five animals were sacrificed on PND 90 by intracardiac perfusion using 10% neutral buffered FA solution. The Cavalieri principle of stereological approaches was used to determine the volume of the DG in these sections. The optical fractionator counting method was used to estimate the total number of granule cells in the DG. The appearance of granule cells was normal under light microscopy in all PND 30 and PND 90 groups. There were significant age-related reductions in the volume of the DG at PND 90 irrespective of which group was examined. Significant age-related neuron loss was also determined at PND 90 compared to that at PND 30. Rats treated with a high concentration FA were found to have fewer granule cells than either the animals treated with a low concentration FA or the control group (p < 0.01 and p < 0.01, respectively) at PND 90 but not at PND 30. These findings clearly indicate that granule cells in the DG may be vulnerable to stress and the concentration of FA to which they are exposed during early postnatal life, and also that a neurotoxic effect of high dose FA on cell number is only seen after a long time period. These results may explain why some disorders do not appear until later life.

Introduction

Formaldehyde (FA), a member of the aldehyde family and one of the simplest organic molecules, is a bactericidal agent and tissue preservative (Bernstein et al., 1984, Naya and Nakanishi, 2005, Yamato et al., 2005). It is found in nature, domestic air, cigarette smoke, and the polluted atmosphere of cities (Bernstein et al., 1984, Songur et al., 2003). In particular, children living in an environment with a high concentration of FA may show signs of disturbance since FA is metabolized in the body as a toxic substance (Bernstein et al., 1984, Naya and Nakanishi, 2005, Yamato et al., 2005). Furthermore, it is widely used in industrial and medical settings. Employees, especially the histologist, anatomist, pathologist and medical students following dissection lectures, are the subjects most frequently exposed to FA gas. FA in excess of certain doses is accepted as being toxic, and its harmful effects increase under room temperature due to easy evaporation and also metabolism into formic acid (Bernstein et al., 1984, Kilburn et al., 1987, Malek et al., 2003, Songur et al., 2003, Gurel et al., 2005, Yamato et al., 2005). Numerous studies have shown that long-term use of FA may cause some disorders related to neurotoxicity (Kilburn et al., 1987, Kilburn, 1994). In this regard, animal models of FA exposure have provided valuable information on its neurotoxic effects (Kilburn, 1994, Sorg and Hochstatter, 1999, Pitten et al., 2000, Songur et al., 2003). In some experiments of a rat model of FA inhalation, FA-exposed animals showed a pronounced impairment in open field, maze trail and central nervous system (CNS) function (Kilburn, 1994, Pitten et al., 2000, Malek et al., 2003). It has been reported that FA may be found in the cerebrospinal fluid and thus affects the neuroglia and nerve cell because it can pass easily through the blood brain barrier (Malek et al., 2003).

The hippocampal formation located in the temporal lobe is a widely investigated anatomical region composed of the entorhinal cortex, the hippocampus itself, the subicular complex and the dentate gyrus (DG). Anatomically, DG has a toothy appearance and is composed of three distinct layers: (1) the molecular layer, continuous with the hippocampus in the hippocampal fissure; (2) the granular layer, consisting of closely arranged spherical or oval neurons called granule cells, whose axons pass through the polymorphic layer ending on the dendrites of pyramidal cells in the hippocampus; and (3) the polymorphic layer (Amaral and Witter, 1989). Of these layers, the principal cell layer of DG is the granule cell layer (Ohnuma and Harris, 2003, Malberg, 2004). It is believed that the hippocampal formation is involved in the control of some behavioral and cognitive functions, including spatial learning (Lemaire et al., 2000, Miki et al., 2000, Eyre et al., 2003). Cell birth and maturation into neurons in the DG are seen throughout the life of animals and humans (Gould et al., 1997, Eriksson et al., 1998, Hastings et al., 2001, Eriksson and Wallin, 2004, Malberg, 2004). Neurogenesis involves the proliferation, migration and differentiation of progenitor cells in the CNS (Nixon and Crews, 2002, Watts et al., 2005). It has been shown that newly generated cells in the adult hippocampus have a neuronal morphology and function (Van Praag et al., 2002, Kempermann et al., 2004). Therefore, it is suggested that adult-generated granule cells in DG are involved in a role of hippocampal-dependent learning (Lemaire et al., 2000, Hastings et al., 2001).

Accordingly, it is possible that FA exposure may cause various morphological changes in the rat brain, but the question that such behavioral deficits are accompanied by morphological alterations in the hippocampal formation of these animals has not been clearly answered to date. In this study, we have used stereological techniques to investigate the effects of FA exposure on hippocampal neurons in the DG of rats at postnatal day (PND) 30 and PND 90. It is well documented that granule cells are the principle neurons of the DG and that they receive fibers from the entorhinal cortex and commissural projections from contralateral mossy cells (Hastings et al., 2001). Thus, an experiment investigating exposure to FA in early postnatal life may give some useful basic information on the development of the brain as well as of the DG.