Subsurface cistern (SSC) proliferation in Purkinje cells of the rat cerebellum in response to acute and chronic exposure to paint thinner: A light and electron microscopy study

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Abstract

Intentional inhalation and occupational exposure are two ways humans are exposed to thinner, a widely employed solvent in industry. Inhalation of thinner induces toxic effects in various organs, with the cerebellum being one of the most affected structures of the CNS. The aim of this work was to describe specific structural alterations of cerebellum Purkinje cells in rats following exposure to thinner for 16 weeks. A histological analysis of the cerebellum of solvent-exposed rats revealed swollen Purkinje cell dendrites surrounded by empty space, and electronic microscopy showed an increase in the number of subsurface cisterns (SSCs) within their dendritic processes. After a period of non-exposure, the number of SSCs decreased without reaching normal levels, suggesting a degree of plasticity. Purkinje cell SSCs, which are derived from smooth endoplasmic reticulum, contain inositol trisphosphate receptors (IP3Rs), ryanodine receptors (RR), and a recently identified characteristic cluster of large conductance calcium-activated potassium (BKCa) channels. We found that SSCs in Purkinje cell dendrites were closely associated with mitochondria, and immunofluorescence microscopy showed higher levels of RR and calbindin receptors (CB), in Purkinje cells of exposed than normal rats. These changes are probably related to behavioral manifestations of cerebellar alterations, such as imbalance and ataxia, consistent with the suggested involvement of increases in SSCs in ataxia in rats and humans. This increase in SSCs, taken together with the localization of RR, IP3R and BKCa proteins in this structure, suggests altered intracellular calcium-buffering processes in the Purkinje cells of thinner-exposed rats.

Introduction

Organic solvents are ubiquitous in the environment and are essential for industry. They are found in many consumer products, such as paint thinners, cleaning fluids, and glues. The abuse of inhalants as recreational substances has increased dramatically. New users in the USA increased 234% between 1990 and 2001, and the rate of first-time use has increased by 243%. Volatile-substance abuse has been reported in the literature over the past 25 years, and has apparently increased as countries have become more industrialized (Butland et al., 2013). Young people frequently abuse volatile substances, and most are initiated into drug abuse with thinner. This substance is readily available and cheaper than other drugs (Flanagan and Ives, 1994).

Thinner is a well-known neurotoxic agent whose abuse is accompanied by a significant decrease in brain and body weights; microcephaly is a teratogenic effect of thinner inhalation. Thinner inhalation can lead to severe central nervous system (CNS) abnormalities, including cerebellar degeneration, cortical atrophy, and impaired mental and intellectual performance (Ron, 1986, Streicher et al., 1981). In fact, the CNS is considered be the primary target of thinner (Benignus, 1981, Lazar et al., 1983), and the principal mechanism of thinner toxicity is thought to be oxidative stress (Baydas et al., 2003, Martinez-Alfaro et al., 2006, Mattia et al., 1993). Oxidative stress has been related to the presence of “black” neurons (Csordas et al., 2003, Gallyas et al., 1992, Ilhan et al., 2004, Ramirez-Exposito and Martinez-Martos, 1999). An increase in the astrocyte-associated glial fibrillar associated protein (GFAP), a specific marker of glial reactivity, has been found in the hippocampus, cortex and cerebellum following thinner inhalation, and is linked to oxidative stress (Baydas et al., 2003).

Animal studies have confirmed that exposure to toluene (a principal solvent component of paint thinner), cause neuronal damage and alter cognitive behavior (Huang et al., 1992, Korbo et al., 1996). Like many other solvents, thinner exerts a characteristic set of acute and chronic effects on humans and experimental animals, including changes in sensory, motor, and cognitive functions (Bushnell et al., 1994, Bushnell and Oshiro, 2000, Pryor, 1991). Clinically, the effects of thinner abuse are characterized by euphoria, disinhibition, hallucinations, tinnitus, ataxia, and confusion. Ataxia refers to a loss of balance and motor coordination due to dysfunction of the cerebellum. Toluene, the principal component of thinner, is a neurotoxin that causes cerebellar ataxia (Escobar and Aruffo, 1980).

Cerebellar Purkinje cells are important elements in cerebellar information processing. Purkinje cells receive many afferents signals, which are integrated and relayed through inhibitory synapses in deep cerebellar nuclei. A reduction in the activity of inhibitory Purkinje cells results in hyperexcitability of their targets and is considered an important feature of cerebellar ataxia. The presence of dark neurons among Purkinje cells, tree swelling, and proliferation of subsurface cisterns (SSCs) are the most conspicuous morphological responses of the cerebellum to thinner inhalation. The SSC as a structure was initially described by Engstrom, Rosenbluth, and Takahashi (Engstrom, 1958, Rosenbluth, 1962, Takahashi and Wood, 1970). Within SSCs are localized proteins important for calcium metabolism, including ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs); more recently, clusters of large conductance, calcium-activated potassium (BKCa) channels have been identified in cerebellar Purkinje cell SSCs.

Because the cerebellum is responsible for equilibrium and fine movement regulation, in the current study, we examined cerebellar histology for evidence of damage following exposure to solvent vapors using a rat model. Our results show a proliferation of SSCs and, importantly, an increase in ryanodine and calbindin receptors in this structure. Moreover, a close relationship between SSCs and mitochondria is seen in cerebellar Purkinje neurons following exposure to thinner vapors. The specific objective of the present work was to microscopically study damage to the rat cerebellum subsequent to long-term (18 weeks) inhalation of thinner vapors.

This work continues a series of previously published studies on thinner abuse in a rat model (Martinez-Alfaro et al., 2010, Martinez-Alfaro et al., 2006). The published works contain more detailed information about: exposure conditions, pathological changes and oxidative stress biomarkers during treatment. Treated rats have significantly lower body weight at the end of treatment, in comparison to control rats. Moreover, brain, lungs, kidneys and lymphocytes of rats exposed to thinner showed oxidative stress (MDA elevation, GSH reduction and DNA oxidation).

Section snippets

Animals

Sixty male Wistar rats from our local animal house, weighing from 80 to 100 g at the beginning of the experiment, were divided into three groups (caged in groups of five): control (n = 16), SHAM (n = 16), and solvent exposure (n = 25). All animals had access ad libitum to food (Purina Chow Rodent Diet; Purina, USA) and water, except during periods of exposure to paint thinner vapors. All animal procedures received prior approval of the Animal Care and Experimental Procedures Committee of our institute

Results

Paraffin sections of the right lobe of the rat cerebellum obtained from control and treated rats were stained and analyzed using the Klüber–Barrera method. The parafloccular lobe, which is well developed in the rat and very easy to identify, was also selected for analysis in this study. Cerebellar sections from control (Fig. 1A) and solvent-exposed rats (B and D) are shown in Fig. 1. The Purkinje cell and its dendritic tree are clearly visible and well defined in slices from rats exposed to

Discussion

The aim of this work was to study structural alterations in the cerebellum induced by thinner inhalation in a rat model of thinner abuse. The CNS is one of the structure affected by the neurotoxicity induced by thinner (Carabez et al., 1998). One of the most important alterations found in rat cerebellum was the proliferation of SSCs in the dendritic tree of Purkinje cells. The observed organelles possess three defining characteristics of SSCs, as originally described: (1) they are bounded by a

Funding

Supported in part by UNAM program PAPIIT IN218507-1822.

Acknowledgements

We thank Martín Garcia, Evelyn Flores, and Sergio Maya for technical assistance. We are grateful to Alta Tecnologia en Laboratorios for lending us the FV-1000 microscope and greatly appreciate their support and technical advice. We are also grateful for the technical advice of Abraham Rosas Arellano (INB-UNAM). The CB and Alexa Fluor 488-conjugated donkey anti-goat IgG (A488D-Ab) antibodies were a generous gift from Dr. Ataúlfo Martinez-Torres.

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    These authors contributed equally to this work.

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