Keywords

1 Introduction

Recently there has been a surge to investigate the effects of nanoparticles on biological systems normally present in the environment (1–3). These small sized (1 to 100 nm) particles have novel properties, which can be highly desirable for applications within the commercial, medical and environmental fields (5,6). Most of these nanoparticles are formed from transition metals, e.g., silver, copper, aluminum, silicon, carbon and metal oxides (1,5). Due to their small sizes, these nanoparticles can either easily cross the blood–brain barrier (BBB) and/or produce damage to the barrier integrity by altering endothelial cell membrane function (5,6). Interestingly, in spite of our increased understanding of BBB function, influence of nanoparticles on BBB is still largely unknown (9,13,15).

It is quite likely that nanoparticles when reaching the CNS compartments may induce profound cellular and molecular stress (8) leading to BBB disruption and brain edema formation through a cascade of secondary cellular and molecular events (8,9,12,13). This investigation is focused on the influence of engineered nanoparticles from metals, e.g., Al, Cu and Ag (50–60 nm) on the BBB permeability to protein tracers, brain edema formation and cell injury in a rat model.

2 Materials and Methods

2.1 Animals

Experiments were carried out on Sprague Dawley rats (body weight 250 to 350 g) housed at controlled ambient temperature (22 ± 1°C) with 12 h light and 12 h dark schedule. Standard laboratory diet and tap water were supplied ad libitum before and after the experiments. All animal experiments described in this review were conducted according to National Institute of Health (NIH), United States Government guidelines for care, handling and maintenance of animals and approved by the Local Institutional Ethics Committee for Animal Care and Research.

2.2 Administration of Nanoparticles

Under equithesin anesthesia (3 mL/kg, i.p.), engineered nanoparticles from Copper (Cu), Aluminum (Al), or Silver (Ag) in the size range of 50 to 60 nm (obtained from US Air Force Research Laboratory from Dr Saber Hussain; and commercially procured from IoLiTec Ionic Liquids Technologies, 79211 Denzlingen, Germany). The nanoparticles were suspended in 0.05% Tween 80 in 0.7% NaCl solution (cf 10, 11, 13). This solution was administered intravenously (30 mg/kg), intraperitoneally (50 mg/kg) or used as cortical superfusion (c.s., 20 µg/10 µL). The Tween 80 solution alone when injected into the carotid artery does not produce any brain or spinal cord pathology (see 11,15). The animals were allowed to survive 24 h after the administration of nanoparticles.

2.3 Blood–Brain Barrier Permeability

The blood–brain barrier (BBB) permeability to Evans blue albumin (2% of a sterile solution, 0.3 mL/100 g body weight) and radioiodine tracer ([131]-Iodine, either 100 µCi/Kg, or a minimum of 0.5 million CPM) given intravenously was determined as described earlier (10).

2.3.1 Morphological Investigations

For morphological investigations, the brains were perfused in situ with 4% paraformaldehyde preceeded with a brief saline rinse (12–14), taken out and photographed. Then coronal sections passing through hippocampus, cerebellum and brain stem were embedded in paraffin. About 3 µm thick sections were cut and stained for standard histological stains, e.g., Nissl or Hematoxylin and Eosin and Luxol Fast Blue (13).

2.3.2 Brain Edema Formation and Electrolyte Content

Brain water content was measured from the differences in the wet and dry weight of the samples (10–14). Volume swelling from the differences between brain water content was calculated (see 13). Normally, an increase in 1% water content represents marked edema formation. After obtaining the dry weight, the tissue was processed to determine the Na+ and K+ content according to standard procedures (4).

2.4 Physiological Variables

In some group of animals mean arterial blood pressure (MABP) and blood gases including arterial pH were also examined using standard procedures (10).

2.4.1 Statistical Analysis

ANOVA followed by Dunnet’s test was used to evaluate statistical significance of the data obtained from one control group. A p-value <0.05 was considered significant.

3 Results

3.1 Effects on Nanoparticles on the BBB Permeability

The Ag, Cu and Al nanoparticles altered the BBB to Evans blue albumin and radioiodine in the rats in a highly selective and specific manner. The leakage of Evans blue was seen on the ventral surface of the brain and in the proximal frontal cortex. The dorsal surfaces of cerebellum and the brain stem showed mild to moderate Evans blue staining (Fig. 1). The effect of Al nanoparticles on the BBB function was much less intense compared to Ag and Cu nanoparticles. Intraperitoneal administration of nanoparticles had least influence on BBB disruption (see Table 1).

Fig. 1
figure 1

Shows extravasation of Evans blue on the dorsal (a) and ventral (b) surfaces of rat brain after Ag nanoparticle treatment. The Ag nanoparticle was administered intravenously (35 mg/kg) and the rat is allowed to survive 24 h after injection. Coronal sections of the brain passing through hippocampus (c) and caudate nucleus (d) are also shown. Leakage of Evans blue dye can be seen in various brain regions (arrows). The deeper parts of the brain, e.g. hippocampus, caudate nucleus, thalamus, hypothalamus, cortical layers including pyriform, cingulate, parietal and temporal cortices, showed moderate blue staining. This indicates widespread leakage of Evans blue albumin within the brain after Ag treatment. Bar = 3 mm (modified after 9 and 15)

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Table 1 Effect of nanoparticles on Blood-Brain Barrier (BBB) permeability, brain edema formation and cell changes in rats and mice at 24 h after administration
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Cortical superfusion with nanoparticles resulted in mild to moderate opening of the BBB to protein tracers largely to be seen on the ipsilateral side. However, the cerebellum and dorsal parts of the brain stem showed leakage of Evans blue albumin as well. This effect was most pronounced with Ag and Cu nanoparticles. The Al nanoparticles showed only faint to mild blue staining (Table 1). Intravenous administration of Al, Cu and Ag nanoparticles induced extravasation of Evans blue and radioiodine tracer in different brain areas (Table 1). There was no difference in radiotracer extravasation when the nanoparticles were administrated as a suspension in water or mixed with Tween 80 (results not shown). Tween 80 or NaCl given in equimolar concentration did not induce radiotracer extravasation in any brain regions compared to control group.

3.1.1 Nanoparticles and Edema Formation

Intravenous administration of Cu nanoparticle resulted in mild but significant edema formation in different parts of the cortex compared to the control group. Thus, about 0.5% to 1.2% increase in brain water content was noted in the cingulate, pyriform and temporal cortices. Administration of Al nanoparticle induced mild increase in water content compared to the controls (see Table 1).

Our data further show that Cu treatment increased Na+ content in the sample with a slight decrease in K+ content (Table 1). Ag treatment also altered ion content in the brain in a similar way. Al treatment showed minimum changes in Na+ and K+ contents in the brain compared to the control groups (Table 1).

3.1.2 Nanoparticles and Physiological Variables

Administration of nanoparticles either into the jugular vein or into the femoral artery slowed the heart rate immediately and the respiratory rate was temporarily increased. This effect lasted for about 10 min. However, none of the animals showed gasping. The effect of Cu and Ag nanoparticles were most pronounced on heart rate and respiration compared to Al nanoparticles (Table 1).

At the onset of nanoparticle (Cu and Ag) administration, the mean arterial blood pressure (MABP) was decreased by 20 to 30 torr for about 5 to 8 min that recovered partially, but remained depressed from the pre-injection value even 90 min after the nanoparticle injection. The decline in MABP following Al nanoparticle was least pronounced (Table 1). Arterial PaO2 increased slightly after nanoparticle administration whereas PaCO2 was either unchanged or decreased slightly in some animals. The arterial pH was not affected significantly (Table 1).

3.1.3 Nanoparticles and Morphological Changes in the Brain

Nerve cell damage in several brain regions showing Evans blue extravasation (Fig. 2) was seen following administration of Ag and Cu nanoparticles (Fig. 2). Alterations in glial cell and myelin also occurred (Fig. 2). These neuropathological changes were least affected by Al nanoparticles (Table 1). Normal animals that received saline or Tween 80 did not show any cell changes in the brain (see Table 1).

Fig. 2
figure 2

Shows loss of myelin and nerve cell damage in rats treated with Ag (c, f, h) or Cu (e, g, d) nanoparticles. Luxol fast blue staining was done to check myelin loss in nanoparticle treated rats compared to controls (Cont a, b). Significant loss of myelin was seen in loss of blue-green staining following Ag© and Cu (d) treatment (arrows). The areas devoid of luxol fast blue (*) are clearly seen (c, d). Nissl staining (e, f, g, h) shows dark and distorted neurons in the hippocampus following Cu (e) or Ag (f) treatment. In the cerebral cortex (g, h) Nissl stain showed many dark and distorted neurons following Cu (g) or Ag (h) treatment. Dark neurons (arrows), and loss of nerve cells (*) are clearly seen in the neuropil. Bar = 60 µm (after Sharma et al., 2009, Ref. 15)

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4 Discussion

The present results are the first to suggest that engineered nanoparticles from metals when administered systemically are able to induce breakdown of the BBB permeability, depending on the route of administration and the type of nanoparticles. Thus, administration of Ag and Cu nanoparticles intravenously or superfused over the cortical surface profoundly induced the breakdown of the BBB to protein tracers compared to Al nanoparticles. On the other hand, intraperitoneal administration of nanoparticles was least effective in BBB disruption. These observations suggest that the amount of nanoparticles reaching the cerebral circulation is largely determining their effects on BBB permeability. Obviously, intravenous administration or cortical superfusion exposes the brain microvessels to these nanoparticles more effectively compared to their administration through intraperitoneal route.

Although, the mechanisms by which nanoparticles influence the BBB function are still unclear, it appears that nanoparticles depending on their characteristics may induce cellular or oxidative stress within the brain microvessels (1, 8, 15). Cellular or oxidative stress is known to induce release of various neurochemicals, cytokines and other neurodestructive factors, e.g., lipid peroxidation, generation of free radicals and nitric oxide (see (9–14)). These neurodestructive elements may then act on the cerebral microvessels either from lumen (when the nanoparticles are administered intravenously) or on the abluminal side (in case of cortical superfusion with nanoparticles) to disrupt the endothelial cell membrane permeability allowing intravascular tracers to leak within the brain microfluid environment (13, 15). Our investigation thus further suggested that nanoparticles are able to disrupt both the blood–brain and brain–blood barriers, not reported earlier.

This breakdown of BBB caused by nanoparticles is unrelated to the possible hyperosmotic effects of Al, Ag or Cu in saline or Tween 80 solution (7, 9, 10). This is apparent from the fact that Tween 80 alone of NaCl solution of equimolar concentration did not affect the BBB function. Moreover, selective effects of nanoparticles with least BBB breakdown by Al nanoparticles compared to Ag and Cu also rule out hyperosmotic effects of solutions per se on BBB dysfunction (7).

Breakdown of the BBB to protein tracers, e.g., Evans blue and radioiodine, leads to vasogenic edema formation and subsequent brain damage (12–14). Significant increase in brain water and volume swelling in the areas showing Evans blue leakage induced by nanoparticles is in line with this idea. Alterations in Na+ and K+ content following nanoparticle treatment further support the development of vasogenic edema formation (4). When the brain fluid microenvironment is altered, then various biochemicals, immunological agents, and neurodestructive factors can easily be transported from blood to brain. Entry of these restricted elements into the fluid microenvironment of the brain will thus initiate serious immunological, biochemical, and cellular or molecular stress leading to nerve cell, glial cell and myelin injury (Sharma HS unpublished observation). Obviously, leakage of BBB and exposure of neurons, glial cells and myelin to exogenous serum factors will induce cell reaction in the brain.

5 Conclusion

In conclusion, our observations suggest that engineered nanoparticles when administered systemically are capable to induce BBB disruption, brain edema formation and lead to abnormal cell reactions. These effects of nanoparticles on brain function are most pronounced with Cu and Ag nanoparticles, compared to Al. It remains to be seen whether nanoparticle-induced brain dysfunction is related to dose and size of the nanoparticles. Furthermore, whether this acute exposure of nanoparticles will further enhance early neurodegenerative changes leading to various brain diseases is unclear and currently being investigated in our laboratory.

Conflict of interest statement We have no conflict of interest with any organizations mentioned below.