Transport of Intranasally Instilled Fine Fe2O3 Particles into the Brain: Micro-distribution, Chemical States, and Histopathological Observation
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- Wang, B., Feng, W.Y., Wang, M. et al. Biol Trace Elem Res (2007) 118: 233. doi:10.1007/s12011-007-0028-6
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It has been demonstrated that inhaled fine (d < 2.5 μm) and ultrafine (d < 100 nm) particles produce more severe toxicity than coarse particles. Some recent data support the concept that the central nervous system (CNS) may be a target for the inhaled fine particulates. This work describes initial observation of the transport of intranasally instilled fine ferric oxide (Fe2O3) particles in animal brain. The iron micro-distribution and chemical state in the mice olfactory bulb and brain stem on day 14 after intranasal instillation of fine Fe2O3 particle (280 ± 80 nm) suspension at a single dose of 40 mg/kg body weight were analyzed by synchrotron radiation x-ray fluorescence and x-ray absorption near-edge structure (XANES). The micro-distribution map of iron in the olfactory bulb and brain stem shows an obvious increase of Fe contents in the olfactory nerve and the trigeminus of brain stem, suggesting that Fe2O3 particles were possibly transported via uptake by sensory nerve endings of the olfactory nerve and trigeminus. The XANES results indicate that the ratios of Fe (III)/Fe (II) were increased in the olfactory bulb and brain stem. The further histopathological observation showed that the neuron fatty degeneration occurred in the CA3 area of hippocampus. Such results imply an adverse impact of inhalation of fine Fe2O3 particles on CNS.
KeywordsFine ferric oxide particleIntranasal instillationOlfactory transportationBrainMicro-distributionChemical statePathological observation
Epidemiological studies conducted in Europe and the United States have demonstrated that inhaled fine (d < 2.5 μm) particles are strongly associated with respiratory and cardiovascular system diseases [1–3]. Many toxicological studies over the last 10 years indicate that particles in the ultrafine size range (d < 100 nm) are likely responsible for most of the adverse health effects [4,5]. Recently, interest in the non-pulmonary targets of inhaled particles has been increasing, as some studies have demonstrated that inhaled fine and ultrafine particles could quickly left the lung and deposit in extra-pulmonary tissues [6,7]. Some recent data support the concept that the brain might be another potential target organ for inhaled fine and ultrafine particles [8,9]. The engineered nanoparticles are of the same size as aerosol ultrafine particles. With the development of the nano-industry, people are at significantly elevated health risks of acute fine and ultrafine particle exposure. The investigation of the fate of the inhaled engineered fine and ultrafine particles in extra-pulmonary site is important for estimation their effects in the target organs.
Depending on the size of fine and ultrafine particle, inhaled nanoparticles are more efficient to deposit in the nasal mucous . The primary olfactory neurons in the olfactory mucosa provide a direct contact with the external environment in the nasal cavity, thus, present a route for exogenous substances to enter the brain . Many studies have shown that the olfactory tract is an important access for metal ions and inhaled foreign particles entering the brain bypass the blood-brain barrier [8–12]. In this study, we hypothesized that fine particle uptake in the olfactory epithelium could transport along the olfactory neurons and enter the brain. We selected fine ferric oxide (Fe2O3) particle representing as a kind of mildly soluble material to observe its possible transportation into the brain via olfactory pathway after intranasal instillation.
Iron in the brain has been extensively studied because of its ability to produce neurotoxicity after high dose occupational exposure [13,14]. Abnormal accumulation of iron in the brain has been implicated in a variety of neurodegenerative diseases, including Hallervorden-Spatz, Parkinson’s disease (PD), and Alzheimer’s disease (AD) [14,15]. Some evidence indicates that iron accumulation in brain regions is an initial event that causes neuronal death or is a consequence of some disease processes . Iron as a redox-active transition metal can play a pivotal role in the formation of hydroxyl radicals through the Fenton reaction in vivo. Oxidative stress resulted from increased iron levels in brain and possibly from defects in antioxidant defense mechanisms is believed to associate with neuronal death in those neurodegenerative disorders [14,15]. Therefore, we are extremely concerned about whether the mildly soluble fine or ultrafine iron particles could transport into the brain like other metals via olfactory pathway during high level inhalation. It has been known that the process of olfactory transportation depends on several factors including particle solubility, particle size, the site of deposition, and so forth . Rao et al.  found that FeSO4 aerosol was not readily transported to the brain via the olfactory tract. However, it has not been known yet whether the iron particles could transport into brain when particles deposit in the nasal mucous.
In our earlier pilot study, the significant increase of iron contents in the mouse olfactory bulb (OB) and deep brain regions had been observed after intranasal instillation of fine Fe2O3 particles, which led us to hypothesize that the routes for translocation of fine or ultrafine Fe2O3 particle to the central nervous system (CNS) via the olfactory tract existed. Therefore, a more detailed study was conducted by synchrotron radiation x-ray fluorescence (SRXRF) and x-ray absorption near-edge structure (XANES) on iron micro-distribution and chemical state in the OB and brain stem after intranasal instillation of fine Fe2O3 particle to the mice.
Materials and Methods
Preparation of Fine Fe2O3 Particle Suspension
Healthy CD-ICR male mice of 4 weeks old and 20- to 22-g weight were supplied by the Department of Laboratory Animal Science, Peking University Health Center. The mice were maintained on the commercial pellet diet and deionized water ad libitum and kept in plastic cages in a 20 ± 2°C, 50–70% relative humidity room with a 12-h light/dark cycle. The exposed group mice were instilled intranasally in the right nasal cavity with 40-mg fine Fe2O3 particle per kilogram body weight. The control mice were given by the same volume of 1% sodium carboxy methyl cellulose solution instead.
SRXRF and XANES Study
On day 14 after intranasal exposure, the mice were killed, and the brains were quickly removed. The OB and brain stem were frozen; coronally cut into 80-μm slice, then fixed onto a polycarbonate film, and dried in a clean box. Because of the limitation of experimental time, three exposed and three control samples were studied.
The SRXRF experiment was performed at the Beijing Synchrotron Radiation Facility (BSRF) 3W1A X-ray fluorescence station in the Institute of High Energy Physics, Chinese Academy of Sciences. The beam line from the storage ring (2.5 GeV, maximum current 120 mA) can provide multi-chromatic x-rays of energy range from 3.5 to 35 keV. Considering the limit of detection, the cross-section of the beam irradiating on the sample was adjusted to about 60 × 80 μm by a Pb collimator with two cross-slices. The iron micro-distributions in the right OB and the whole brain stem were studied. The sample platform was moved by a two-dimensional stepping motor. The Fe distribution in the OB was continuously scanned at a step of 150 μm for the X direction and 200 μm for the Y direction. For the brain stem sample, the scan was performed by 300 μm step-size along the X direction and 200 μm along the Y direction. About 200 points were measured for each OB slice and 210 points for brain stem one. Each spot was irradiated for about 60 s. The x-ray spectra were analyzed by the AXIL program, and all x-ray spectra and their Compton scattering intensity were normalized to the collecting time and the counting of the ionization chamber. The relative content of Fe was obtained by the ratio of net x-ray peak area (with subtracting the contribution of the polycarbonate film) to the relative Compton scattering intensity. Then, the micro-distribution map of Fe in the brain region can be obtained.
The XANES spectra were collected at the beam line 1W1B of BSRF. The synchrotron radiation was monochromatized by a Si (111) double crystal monochromator. Harmonic rejection was achieved by detuning the second crystal of the monochromator. Energy resolution was estimated to be about 0.3 eV by the copper foil three-dimensional near-edge feature. The beam size on the sample was 900 × 300 μm2. Energy calibration was monitored using an iron foil and was set as 7.112 keV. Iron K-edge XANES spectra were recorded from 200 eV below to 200 eV above the iron K-edge threshold energy (7.112 keV). The data of FeO and Fe2O3 chemical standards were collected at transmission mode using ion chamber with N2/Ar filled gas. The fluorescence signal of brain samples was detected by a Lytle fluorescence ionization chamber. The absorption coefficients were calculated from the incident (I0) and transmission intensity (I) or fluorescence intensity (If), and the data normalization was carried out by WINXAS 97.
After the mice were killed, the brains were quickly removed and fixed in 10% formalin, and then embedded into paraffin, sectioned for 5-μm thick, and finally mounted on the glass microscope slides using standard histopathological techniques. The sections were stained with hematoxylin–eosin and examined by optical microscopy.
Fe Micro-distribution in the OB and Brain Stem by SRXRF
Fe K-edge XANES Analyses
Absorption Edge, Edge Shift, and Fe (II)/Fe (III) Ratio in the Brain Samples
Absorption edge (keV)
Edge shift (eV)
Fe (II)/Fe (III) ratioa
In our early pilot study, we observed that significantly higher iron existed in the OB and could further transport into other brain regions, such as the brain stem after intranasal instillation. Therefore, to further understand the transport of inhaled fine Fe2O3 particle into the brain, the micro-distribution and chemical states of iron in the OB and brain stem were investigated.
Synchrotron radiation is an ideal x-ray source that provides continuum and high intensity photon for the investigation of trace metallic elements contained in biological specimens. SRXRF and XANES can provide information on the contents and chemical environment of trace elements in biological samples. In the x-ray fluorescence station of BSRF, the detection limit for Fe is about 10–15 g . With high sensitivity, collimation, and good spatial resolution (∼10 μm), SRXRF analysis could provide micro-distribution map of iron in the brain regions.
Neuronal uptake and translocation of nanoscaled solid particle via neuronal axons has been described for more than 60 years [22–24]. However, it has received little attention from toxicologists. Until recently, Oberdörster et al.  reported that inhaled ultrafine particle 13C (36 nm) could transport along axons of the olfactory nerve into the brain, which caused more attention about fine and ultrafine particle impact on CNS. In our experiment, the Fe level in the OB and brain stem slices were found to increase after 2 weeks after a single intranasal instillation of fine Fe2O3 particle, which suggested that iron particle entered CNS. The higher Fe levels in the ON and Gl layers indicated that Fe2O3 particle entered the OB via ON and could transport along the secondary nerve of Gl, where olfactory sensory neurons make contact with OB neurons and contain projections from interneurons. The increased Fe in AOE and lo indicated that Fe was transported to AOE along the axons from the mitral and tufted cells in Gl via lo. Such a conclusion is consistent with the results of inhaled ultrafine carbon particles  and manganese oxide particle . Oberdörster et al.  found significant and consistent increases of 13C in the OB throughout the 7-day postexposure period after the 6-h inhalation exposure to the ultrafine elemental 13C particles. Fechter et al.  demonstrated that small particle MnO2 (1.3 μm) resulted in an elevation of OB manganese concentration. Lee et al.  found the bovine herpesvirus type 5 occurred in the areas of MOB and AO after intranasal inoculation.
The higher Fe levels in the reticular formation and spinal trigeminal regions of the brain stem indicated that iron was transported to deeper brain compartments like soluble Mn via trigeminus  after nasal instillation. The increase of iron levels in the brain stem has been demonstrated to be closely related with various neurodegenerative diseases, such as PD and AD [13,15]. The reticular formation is the central core of brain stem, connecting with all parts of the brain and brain stem, and is critical for the maintenance of consciousness . The increased iron level in the reticular formation indicated that Fe entered the parenchyma portion of the brain stem. Trigeminal nerve is composed of three large branches including ophthalmic (V1, sensory), maxillary (V2, sensory), and mandibular (V3, motor and sensory) branches, and the major sensory innervation to the nasal cavity is supplied by sensory nerve endings of V1 and V2 branches of the trigeminal nerve . Some of the trigeminal sensory nerve endings in the nasal epithelium also have branches directly reaching the OB . Trigeminal nerve root connects the brain stem, and trigeminal nerve nucleus extends through the whole brain stem, midbrain, and medulla. Thus, based on the results of the increased Fe in the trigeminus region of brain stem, we suspect that the increased Fe in the brain stem was possibly transported via sensory nerve endings of trigeminus. Such a delivery route by trigeminal uptake of inhaled metals to enter the CNS has already been demonstrated by the study of inhaled manganese chloride in rats and mice . However, the reasons for the disturbance of Fe distribution in the brain stem are unknown and need further investigation.
The mechanism of iron transport across blood–brain barrier has not yet been completely clarified. The popular accepted suggestion is that the transferrin–transferrin receptor (Tf–TfR) is the major route for iron transport across the luminal membrane of the capillary endothelium . The iron possibly crosses the luminal membrane and enters the interstitial fluid in the form of Fe2+ . The Tf–Fe and non-Tf-bound iron will be rapidly transported in the rat brain for several hours after administration of soluble iron ions . But in this study, the elevated Fe content in the OB was observed even on the postexposure day 14 after a single dose of Fe2O3 particle intranasal instillation. Therefore, as the results, the Fe2O3 particle was suggested transport via olfactory pathway. Nevertheless, this result is different from the observation of Rao et al. , who demonstrated that FeSO4 aerosol (2.99 μm) could not enter the brain via olfactory pathway. The different results might be because of the different size and chemical composition of the two kinds of iron particles. The size of the FeSO4 aerosol in Rao’s study was ten times of Fe2O3 particle used in this study; thus, the size difference might contribute to the different results. Fechter et al.  demonstrated that small particle aerosol deposited in the head airways taken up by olfactory nerve and transported to the OB was much more efficient than large ones.
The final chemical speciation of iron after iron particle deposition in CNS may highly associate with iron physiological function and potential neurotoxicity. The questions whether the iron particles maintain their particulate state in the deposition site or to be dissolved before translocation, during translocation, or after deposition in the destination need further study. XANES is based on the sensitivities of x-ray absorption to the valence state and neighboring atoms, which can give both structural and electronic configuration information . The absorption edge energy (E0) depends on the valence of absorption atom and could slightly shift caused by the variation of the effective atomic charge . As iron in the brain is a mixture of Fe (II) and Fe (III), FeO and Fe2O3 were used to represent the two major valent states of iron. Such an assumption might be too simple to represent the complex speciation of iron in the brain. However, this assumption may be justified if the chemical state of the deposited Fe2O3 particle was only concerned about. The Fe K-edge XANES measurement showed that the E0 values in the OB and brain stem of the exposed mice shift to the values of Fe2O3. Furthermore, the elevated ratios of Fe (II)/Fe (III) in the exposed mice demonstrated that Fe (III) increased in the OB and brain stem.
Our recent results show that the Fe level in the hippocampus has increased on day 14 after intranasal exposure (data not shown here). This is in accordance with the histopathological observation at the same time point. The results demonstrated that the oxidative damage might occur in hippocampus.
In conclusion, the results demonstrated that the transportation route of inhaled Fe2O3 particle into the brain may be via the olfactory route or trigeminal pathway. The XANES measurement indicated that Fe (III) level in the OB and brain stem of the iron-exposed mice significantly increased compared to the controls. An important finding is that the neuron fatty degeneration in CA3 area of the hippocampus occurred in the 14th day after intranasal instillation of fine Fe2O3 particles.
Although there are enormous differences in nasal mucosa between rodent animals and human beings, the results may call attention to the workers at occupational potential risk for exposure to the fine iron solid particle.
The authors are grateful to the foundation of National Basic Research Program of China (2006CB705605), the Chinese Academy of Sciences, and the National Natural Science Foundation of China (10490180, 20475055, 10675139).