In several neuropathological conditions, microglia can become overactivated and cause neurotoxicity by initiating neuronal damage in response to pro-inflammatory stimuli. Our previous studies have shown that exposure to electromagnetic fields (EMF) activates cultured microglia to produce tumor necrosis factor (TNF)-α and nitric oxide (NO) through signal transduction involving the activator of transcription STAT3. Here, we investigated the role of STAT3 signaling in EMF-induced microglial activation and pro-inﬂammatory responses in more detail than the previous study.N9 microglial cells were treated with EMF exposure or a sham treatment, with or without pretreatment with an inhibitor  of the Janus family of tyrosine kinases (JAK). The activation state of microglia was assessed via immunoreaction using the microglial marker CD11b. Levels of inducible nitric oxide synthase (iNOS), TNF-α and NO were measured using real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and the nitrate reductase method. Activation of JAKs and STAT3 proteins was evaluated by western blotting for specific tyrosine phosphorylation. The ability of STAT3 to bind to DNA was detected with an electrophoresis mobility shift assay (EMSA).EMF was found to significantly induce phosphorylation of JAK2 and STAT3, and DNA-binding ability of STAT3 in N9 microglia. In addition, EMF dramatically increased the expression of CD11b, TNF-α and iNOS, and the production of NO. P6 strongly suppressed the phosphorylation of JAK2 and STAT3 and diminished STAT3 activity in EMF-stimulated microglia. Interestingly, expression of CD11b as well as gene expression and production of TNF-α and iNOS were suppressed by P6 at 12 h, but not at 3 h, after EMF exposure.EMF exposure directly triggers initial activation of microglia and produces a significant pro-inflammatory response. Our findings confirm that the JAK2-STAT3 pathway may not mediate this initial microglial activation but does promote pro-inflammatory responses in EMF-stimulated microglial cells. Thus, the JAK2-STAT3 pathway might be a therapeutic target for reducing pro-inflammatory responses in EMF-activated microglia. In vivo animal experiments involving microglial activation, however, cannot clearly explain whether such activation is induced directly by EMF or indirectly as a consequence of neuronal injury from EMF exposure.The rapid development of society and technology has led to an unprecedented increase in the number and diversity of sources of electromagnetic fields (EMFs), including power lines, electric appliances, radio transmitters, and microwave sources. Numerous studies have investigated the effects of occupational or residential exposure to EMF, which has been identified as the fourth largest pollution hazard -7. SeverMicroglia, the resident innate immune cells in the CNS, become activated in response to certain cues, such as brain injury and immunological stimuli ,21. Actii.e., NF-κB, AP-1 and C/EBP, are involved in microglial activation in vivo and in vitro : forward 5'-TAAAGACCTCTATGCCAACACAGT-3'; reverse 5'-CACGATGGAGGGGCCGGACTCATC-3'. iNOS [GenBank: 010927.2]: forward 5'-AACTGTAGCACAGCACAGGAAA-3'; reverse 5'-ACAAGATCAGGAGGGATTTCAA-3'. TNF-α [GenBank: 013693.2]: forward 5'-GCCTATGTCTCAGCCTCTTCTC-3'; reverse 5'-GGAGGTTGACTTTCTCCTGGTA-3'. The amplification reaction was carried out in a Perkin-Elmer GeneAmp . The resulting PCR products were separated in 1.5% agarose gels and visualized with ethidium bromide staining. For semi-quantitative evaluation, densitometric analysis was performed using Quantity One software .Total RNA was isolated using Trizol  from 6-well plates according to the manufacturer's protocol. The integrity of the RNA sample was confirmed with gel electrophoresis and by reverse transcription-PCR using primers for house-keeping genes. Moloney murine leukemia virus (M-MLV) reverse transcriptase  was used to convert 1 μg of total RNA into cDNA at 42°C. The RT-PCR exponential phase was determined to be 20-32 cycles for semiquantitative comparisons. β-Actin was measured as a loading control. The amplification of cDNA was performed with the following primers: Data are presented as the mean of each treatment group ± standard deviation of the mean (SD). Statistical differences between the groups were assessed by one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range test. Statistical significance was established at P < 0.05 unless otherwise indicated.It has been previously suggested that activated microglia express different proteins and surface markers. Of these, CD11b has the greatest biological significance ,64. BecaGiven the pro-inflammatory effect of EMF exposure on microglia, we measured levels of TNF-α and iNOS, and the resulting NO production, in cell culture medium supernatants at the indicated times after EMF exposure. As shown in Figure In previous work, we have shown that activation of JAKs and STAT3 is involved in EMF-activated microglia . To furtUnder basal conditions, STATs are located in the cytoplasm; however, when these transcription factors become phosphorylated, they translocate to the nucleus within minutes . Accordii.e., the JAKs [Given the above results, we focused our studies on the upstream tyrosine kinases of the STAT3 signaling molecule, the JAKs , and perTo further assess the potential role of the JAK2-STAT3 pathway in EMF-induced activation and pro-inflammatory responses of N9 microglia, we examined whether the JAK inhibitor P6 could affect EMF-induced increase of TNF-α, iNOS and NO, and the initial activation of microglia. Western blot analysis and EMSA experiments show that P6 preconditioning completely blocks activation of JAK2 and STAT3 at 3 and 12 h after EMF exposure Figure  &5. Our In the present study, we observed N9 microglial activation and pro-inflammatory responses after EMF exposure. We found that the JAK2-STAT3 pathway is activated in EMF-stimulated N9 microglial cells. The activation of microglia and the secretion of pro-inflammatory factors were significantly reduced by P6 treatment at 12 h after EMF exposure, while P6 preconditioning did not inhibit the above processes at 3 h post exposure. Our data suggest that the JAK2-STAT3 pathway may play a pivotal role in the pro-inflammatory response but not in the initial activation of microglia after EMF exposure.in vitro model exposing N9 cells to 2.45 GHz using a waveguide system that simulates occupational or residential exposure, at a specific absorption rate (SAR) of 6 W/kg. This result suggests that EMF may potentially affect microglial activation. Our data are consistent with earlier findings demonstrating increased glial reactivity in a model using about 900 MHz from a global system for mobile communication, at a high SAR of 6 W/kg [It has been reported that increased CD11b expression corresponds to extent of microglial activation ,64,68. Hf 6 W/kg ,18,69. Of 6 W/kg -72. Takef 6 W/kg , could pMicroglia are known to be exquisite sensors of even minor pathological changes in the CNS ,61. TheyIt is well known that microglia monitor the external environment  and respTo investigate the potential function of the JAK-STAT pathway in EMF-activated microglia, we next examined whether the JAK inhibitor P6 could affect the EMF-induced increases of TNF-α, iNOS, NO and CD11b. P6 can effectively block the activation of JAK1, JAK2 and STAT3 ,88. Our et al.[et al. [Most previous studies have shown that the JAK-STAT signaling pathway is involved in microglial activation -55. In oet al. reported.[et al.  reportedet al. [et al. [It has been demonstrated that activated microglia secrete a diverse range of pro-inflammatory and neurotoxic factors such as superoxide, TNF-α, interleukin (IL)-1β, IL-6 and NO . The cytet al.  reported [et al.  indicateWe studied the effects of EMF exposure on cultured N9 microglial cells and demonstrate that an initial activation of microglia is induced by EMF exposure. In addition, many other physical factors such as infrasound exposure, irradiation, heat shock treatment and hyperthermia, can stimulate activation and pro-inflammatory reactions of microglia -100. TheOur results strongly suggest that external electromagnetic emission as a physical stimulation directly triggers an initial activation of microglia and produces significant pro-inflammatory responses. Activation of JAK2-STAT3 signaling occurs in parallel with the microglial activation and the release of pro-inflammatory factors. Microglial activation and pro-inflammatory responses are significantly reduced by P6 at 12 h, but not at 3 h, after EMF exposure. These results suggest that the JAK2-STAT3 pathway may not mediate the initial microglial activation, but does promote pro-inflammatory responses in EMF-stimulated microglial cells. Our results provide a basis to determine whether the pro-inflammatory responses of EMF-stimulated microglia can be suppressed by inhibition of the JAK2-STAT3 pathway in therapeutic interventions.EMF: Electromagnetic fields; NO: nitric oxide; TNF-α: tumor necrosis factor-α; JAK-STAT: Janus kinase-signal transducers and activators of transcription; iNOS: inducible NO synthase; P6: Pyridone 6; FACS: fluorescence activated cell sorting; EMSA: electrophoresis mobility shift assay; ELISA: enzyme-linked immunosorbent assay.The authors declare that they have no competing interests.This study is based on an original idea of XSY. XSY and GLH wrote the manuscript. XSY and MQL carried out the FACS and confocal double-label immunofluorescence assays. GLH, YTH, CHC carried out the RT-PCR, western blotting and EMSA assays. YW carried out some mediator assays. GBZ provided EMF exposure system. All authors have read and approved the final manuscript.


To apply a scaled, preference-based measure to the evaluation of health-related quality of life (HRQoL) in Parkinson's disease (PD); to evaluate the relationship between disease-specific rating scales and estimated HRQoL; and to identify predictors of diminished HRQoL.Scaled, preference-based measures of HRQoL ("utilities") serve as indices of impact of disease, and can be used to generate quality-adjusted estimates of survival for health-economic evaluations. Evaluation of utilities for PD and their correlation with standard rating scales have been limited.Utilities were generated using the Health Utilities Index Mark III (HUI-III) on consecutive patients attending a PD Clinic between October 2003 and June 2006. Disease severity, medical, surgical ), and demographic information were used as model covariates. Predictors of HUI-III utility scores were evaluated using the Wilxocon rank-sum test and linear regression models.2 = 0.56, P < 0.001) was highly predictive of HRQoL. UPDRS-III was a weaker, but still significant, predictor of utility scores, even after adjustment for UPDRS-II (P = 0.01).68 men with a diagnosis of PD and a mean age of 74.0 (SD 7.4) were included in the data analysis. Mean HUI-III utility at first visit was 0.45 (SD 0.33). In multivariable models, UPDRS-II score (rPoor self-care in PD reflected by worsening UPDRS-II scores is strongly correlated with low generic HRQoL. HUI-III-based health utilities display convergent validity with the UPDRS-II. These findings highlight the importance of measures of independence as determinants of HRQoL in PD, and will facilitate the utilization of existing UPDRS data into economic analyses of PD therapies. Parkinson's disease (PD) is a chronic neurodegenerative illness that results from progressive cell death affecting movement, mood, cognition and autonomic function . The preThe precise effect of optimal PD treatment on life expectancy is unclear, but living with this chronic degenerative illness is thought to have a profound negative impact on health-related quality of life (HRQoL) due to both disease manifestations, and the adverse effects of medical and surgical management strategies -9. As suThe Unified Parkinson Disease Rating Scale (UPDRS) consists of assessments in 4 domains including, mood and cognition (UPDRS I), activities of daily living (UPDRS II), motor symptom severity (UPDRS III) and complications of treatment (UPDRS IV) ; it is tGiven the increasing awareness of HRQoL as an important end-point that may not correlate directly with physical disability, there has been a growing literature documenting the predictors of low HRQoL in PD -17. HoweThe study population consisted of individuals attending the Philadelphia Veterans Administration Parkinson's Disease Research, Education and Clinical Center (PADRECC) between October 2003 and June 2006 with an ICD-9 diagnosis of Parkinsonism or PD. The PADRECC is a multidisciplinary center providing subspecialty care to veterans with PD and other movement disorders and serves a catchments area that covers Pennsylvania, New England and the Mid-Atlantic States. The population of veterans receiving medical care through the Veterans Administration healthcare system in this area is 998,061, of whom approximately 5303 have diagnosed PD. Individuals from this cohort are referred to PADRECC for expert guidance on disease management. Charts of all patients attending the PADRECC during the study period were reviewed. As this was a longitudinal prospective cohort study with respect to the outcome of interest (HUI-III), only individuals with at least 2 completed HUI-III questionnaires (from 2 separate visits) were eligible for inclusion. Review of the diagnosis of parkinsonism was further scrutinized and only individuals fulfilling United Kingdom Brain Bank Criteria  for idioHealth utilities are scaled, preference-based generic measures of health-related quality of life that lie on a zero-to-one scale, with a utility of 1 equivalent to perfect health, and 0, equivalent to death. . While utilities can be elicited using "standard-gamble" or "time-tradeoff" methods, these are intellectually rigorous, and may be upsetting to study subjects . The useP < 0.15 level were considered candidate covariates in multivariable regression models. Multivariable models were constructed using a stepwise selection algorithm, with covariates retained for P < 0.15 [We performed both cross-sectional analyses on baseline data collected for the study cohort, and longitudinal analyses in which we evaluated change in utility scores over time. Baseline HUI-III-based utility scores were evaluated for the cohort as a whole using descriptive statistics. The relationships between UPDRS scores and raw and log-transformed HUI-III utilities were assessed graphically. We evaluated the association between baseline patient characteristics (including PD severity) and baseline HUI-III scores through construction of bi-variable least-squares regression models, with standard errors adjusted to account for multiple measurements on some study subjects. Characteristics that were associated with HUI-III scores at the P < 0.15 . We creaP < 0.15 . Interacand UPDRS scores were available, we further explored the relationship between change in HUI-III scores and UPRDS III scores using the approach of Fitzpatrick et al. [Longitudinal changes over time in HUI-III scores, and UPDRS scores, were evaluated using repeated-measures ANOVA. For the subset of individuals (N = 20) for whom repeated HUI-III k et al. , with caWe screened 156 consecutive patients assessed for parkinsonism in our clinic over the study period. Of these 88 (57%) had more than 1 evaluation of health-related quality of life, and so were included in the study. Of these individuals, 20 had parkinsonism but did not meet Brain Bank criteria for PD; among excluded individuals six were diagnosed with likely vascular parkinsonism; eight were excluded based on atypical features not suggestive of idiopathic Parkinson's disease, two each were excluded based on diagnoses of multisystem atrophy and suspected diffuse Lewy body dementia, and one each was excluded based on diagnoses of fronto-temporal dementia, and progressive supranuclear palsy.Baseline patient characteristics are outlined in Additional File Comorbid medical conditions identified in the cohort included coronary artery disease, stroke, arthritis and diabetes mellitus. On average subjects had disease duration of 8 years at the time of the first recorded visit, with moderate disease severity (reflected by an average UPDRS III score of 30 and H+Y score of 2.8). Mean dosage of anti-parkinsonian medications, expressed as levodopa equivalent dose (LED) [The average value for baseline HUI-derived utility weights was 0.42 range -0.15 to 1.0). In univariable regression models, stroke was significantly associated with reduced HUI-derived utility weights; borderline significant associations were seen with diabetes and marital status  and S+E scores were independent predictors of HRQoL; increased disease duration was associated with The average time interval between first and last assessment in the cohort was 6.6 months (SD 4.9). The mean reduction in HUI-III utilities between first and last assessment was 0.014 (SD 0.25); 34 individuals (50%) experienced a net reduction in utility, 33 (49%) experienced a gain in utility, and 1 (1%) had no change in health utility. When utilities were analyzed using repeated measures ANOVA, there was no reduction in utility scores with succeeding visits (P = 0.67). Significant changes were identified in Schwab and England scores (P = 0.02), but not in UPDRS-III scores (P = 0.66) or Hoehn and Yahr scores (0.11) using a similar approach. Repeated measurements of UPDRS-II scores were obtained in only 20 of 68 subjects; there was no significant change over time in these scores (0.50).Notwithstanding the small number of individuals with both repeated HUI-III and UPDRS measurements, significant Spearman correlations were identified between changes in HUI-III scores  and rescaled change in UPDRS-III scores , Schwab and England scores , and Hoehn and Yahr scores . The largest correlation coefficient was observed for rescaled change in UPDRS-II scores, though because of the small numbers of individuals with repeated UPDRS-II measurement this was not statistically significant . In a multivariable regression model, changes in HUI-III utilities were predicted only by changes in UPDRS-III scores  and time between first and last evaluation .Although Parkinson's disease is most prominently identified with physical symptoms such as tremors and akinesia, this disease has a substantial impact beyond motor impairment and physical disability with an on overall reduction in all health-related quality of life dimensions including social and emotional well-being. To date, the relatively limited application of existing tools for the measurement of health-related quality of life (HRQoL) has made it difficult to compare the loss of HRQoL in PD to that experienced by individuals with other chronic conditions . Using aWe are aware of at least one other prior effort to map health utilities onto UPDRS scores ; SiderowIn comparison to the Siderowf study, our study further refined the relationship between health utilities and UPDRS scores. Perhaps surprisingly, we found that these reductions were most strongly correlated with the self-care component of the UPDRS (UPDRS-II), rather than the UPDRS-III motor sub-score. This finding serves as an important reminder that loss of independence may be an important source of morbidity in individuals with PD. As we demonstrated in regression analyses Figure , the corOther important predictors of low baseline HRQoL in this study included reductions in S+E disability scores, and higher axial sub-scores (PIGD). Though health-related quality of life and self-care ability in PD are inextricably linked to severity of motor dysfunction, the relationship between motor impairment and reduction in health-related quality of life may be complex and indirect, as demonstrated by our failure to find an independent relationship between UPDRS motor III sub-scores and HUI, after controlling for UPDRS-II and other scores. These results are consistent with previous findings that motor impairment in and of itself does not reduce health-related quality of life but the functional consequences of poor motor function including loss of self-care capabilities, inability to ambulate and loss of independence and its emotional consequences that may provide the link between physical impairment and low HRQoL ,35.We failed to find an association between either cognitive impairment or evidence of depression and low HRQoL, similar to one other study . HoweverSix prior longitudinal studies have evaluated HRQoL in PD. The first, based on a community-based cohort, found no relationship between any baseline clinical characteristics and reduction in HRQoL . AnotherForsaa et al.  prospectMarras et al. also evaluated predictors of diminished HRQoL  using a Health utility estimates and most indices of PD severity were relatively stable over the course of our study, which may reflect the relatively short duration of study, and perhaps also the fact that notwithstanding the decline in status expected with a degenerative disease like PD, at least some subjects may have experienced improved health-related quality of life as a result of optimized medical management following referral to the PADRECC. Changes in utility were correlated with changes in multiple PD-specific measures, though our ability to document relationships between changes in health-related quality of life and changes in UPDRS-II scores were limited by the fact that repeated UPDRS-II scores were available in only a small subset of subjects.This study had several important limitations. Our failure to identify a link between depression and low HRQoL contrasts with the results of other studies ,38-45 anOther limitations of this study relate to the generalizability of findings in a cohort of male U.S. veterans: our findings may not be generalizable to non-veterans or to women, as they were not represented in our cohort. Previous epidemiological surveys have suggested gender differences in PD; Men have been described to have earlier symptom onset , increasIn conclusion, we sought to evaluate health-related quality of life in PD using a "health utilities index" approach, and to assess the relationship between health utility scores and PD severity as measured using standard disease-specific tools. In cross-sectional analyses, we identified ADL-related components of the UPDRS as most closely linked to health-related quality of life, a finding that underscores the fact that PD manifests in dimensions aside from movement and motor control. Our findings, although preliminary, may pave the way for translation of PD-specific measures of disease severity into health utility scores, particularly if our findings can be replicated and externally validated in other populations and by other investigators.GKF was responsible for study conception, development of the study protocol, data collection and analysis. She wrote the first draft of the manuscript and revised the manuscript for important intellectual content. MBS was responsible for study conception, contributed to the development of the study protocol, and revised the manuscript for important intellectual content. DNF contributed to development of the study protocol, and data analysis, and revised the manuscript for important intellectual content. All authors have seen and approved the final manuscript draft.The authors declare that they have no competing interests. GFK had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysisTable S1: Characteristics of PD patients at the Philadelphia PADRECC at First Visit and Relationship with Health-Related Quality of Life in Univariable Regression ModelsClick here for file


One particularly exciting field of research involves the use of gold nanoparticles (GNPs) in the detection and treatment of cancer cells in the liver. The detection and treatment of cancer is an area in which the light absorption and emission characteristics of GNPs have become useful. Currently, there are no data available regarding the fluorescence spectra or in vivo accumulation of nanoparticles (NPs) in rat liver after repeated administration. In an attempt to characterise the potential toxicity or hazards of GNPs in therapeutic or diagnostic use, the present study measured fluorescence spectra, bioaccumulation and toxic effects of GNPs at 3 and 7 days following intraperitoneal administration of a 50 μl/day dose of 10, 20 or 50 nm GNPs in rats.The experimental rats were divided into one normal group (Ng) and six experimental groups . A 50 μl dose of GNPs (0.1% Au by volume) was administered to the animals via intraperitoneal injection, and fluorescence measurements were used to identify the toxicity and tissue distribution of GNPs in vivo. Seventy healthy male Wistar-Kyoto rats were exposed to GNPs, and tissue distribution and toxicity were evaluated after 3 or 7 days of repeated exposure.After administration of 10 and 20 nm GNPs into the experimental rats, two fluorescence peaks were observed at 438 nm and 487 nm in the digested liver tissue. The fluorescence intensity for 10 and 20 nm GNPs (both first and second peaks) increased with the infusion time of GNPs in test rats compared to normal rats. The position of the first peak was similar for G1A, G2A, G1B, G2B, G3B and the normal (438 nm); that for G3A was shifted to a longer wavelength (444 nm) compared to the normal. The position of the second peak was similar for G1A, G1B, G2A, G2B and the control (487 nm), while it was shifted to a shorter wavelength for G3A (483 nm) and G3B (483 nm). The fluorescence intensity of the first and second peaks increased for G1A, G2A, G1B and G2B, while it decreased for G3A and G3B compared to the control.The fluorescence intensity of GNPs varied with the number, size and shape of particles and with the ratio of surface area to volume in a given sample. Fluorescence intensity changes during infusion depended on the size and shape of GNPs, with smaller particles experiencing larger changes during the infusion time in addition to the quenching produced by the larger GNPs. It is likely that smaller particles, which have a much higher ratio of surface area to volume compared to larger particles, are more prone to aggregation and surface interaction with biological components. This study suggests that fluorescence intensity can be used to evaluate bioaccumulation and the toxicity of gold nanoparticles in rats. Nanoparticles (NPs) offer great promise for biomedical applications, particularly in pharmaceutical delivery and novel diagnostic and therapeutic methods .Despite the many potential therapeutic benefits of nanoparticles, some studies indicate that certain nanoparticles, due to their small size and unique physical properties, may cause adverse effects ,3. The sThe small size of NPs may enable interactions with specific biological targets. Furthermore, metallic NPs can be made to exhibit a resonant response to a time-dependent magnetic field, producing a potentially useful energy transfer to the particles ,6. ThereGold NPs (GNPs) show several features that make them well suited for biomedical applications, including straightforward synthesis, stability, and the potential for surface modification with active biological molecules such as peptides or proteins .Semmler-Behnke et al. observed that a considerable percentage of 18 nm GNPs is removed from the blood and trapped predominantly in the liver and spleen .Fluorescence is the emission of light by a substance following absorption of light or other electromagnetic radiation of a different wavelength. In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. However, when the absorbed electromagnetic radiation is intense, it is possible for one molecule to absorb two photons; this two-photon absorption can lead to emission of radiation having a shorter wavelength than the absorbed radiation.The origin of the unique optical properties of GNPs is a phenomenon known as surface plasmon resonance (SPR). The exposure of nanoparticles to electromagnetic radiation of a wavelength much smaller than the GNP diameter induces coherent, resonant oscillations of the conduction-band electrons across the nanoparticles. These oscillations are known as the SPR, which lies in the visible frequency range and results in strong optical absorbance and scattering by the GNPs ,12. ThisDespite these many useful applications of NPs, numerous studies have shown that exposure to smaller-sized particles produces greater inflammatory and cytotoxic responses compared to exposures to larger-sized particles at the same mass concentration . It is bThe toxicity of NPs is thought to depend on the size, surface area, composition, and shape of the nanomaterial. Particle size plays a role in how the body responds to, distributes, and eliminates materials ,15; it cThe particle-size-dependent organ distribution of GNPs has been studied in vivo -26. HyllGNPs can be used in various biomedical applications; however, very little is known about the size, dose and exposure dependence of in vivo nanoparticle processing kinetics. Furthermore, there are currently no data available regarding the fluorescence spectra and accumulation of nanoparticles (NPs) in rat liver following repeated in vivo administration.GNPs of different sizes  were purchased. All GNPs used in this study were in aqueous solution at a concentration of 0.01%. The mean size and morphology of these GNPs were evaluated from transmission electron microscope (TEM) images. The high electron density of gold makes this method particularly suitable for evaluating the homogeneity of the nanoparticles in terms of shape and size.Healthy male Wistar-Kyoto rats were obtained from the Laboratory Animal Centre . Rats of age 8-12 weeks (approximately 250 g body weight) were housed in pairs in humidity- and temperature-controlled ventilated cages on a 12 h day/night cycle. A conventional rodent diet and water were provided. Forty rats were individually caged and divided into a control group (NG: n = 10), group 1 , group 2  and group 3 . In addition, histological investigations of 10 rats were performed in support of this study. Doses (50 μl) of 10, 20 or 50 nm GNPs in aqueous solution were administered to the animals via intraperitoneal injection every day for 3 or 7 days. The rats were anesthetised by inhalation of 5% isoflurane until muscular tonus relaxed. Blood and liver tissue were collected from each rat. A blood sample of approximately 1 ml was obtained from each rat via venepuncture of an antecubital vein; the blood was mixed with 0.8 ml of heparin to prevent coagulation. In order to assess tissue uptake, as much blood as possible was collected from the rats to maximise residual blood drainage from the organs. All experiments were conducted in accordance with guidelines approved by the King Saud University Local Animal Care and Use Committee.3 was added. The closed reaction vessel was heated in a 130°C oven until digestion was completed. Samples were then diluted to a final volume of 20 ml with quartz-distilled water and stored in 1 oz. polyethylene bottles for subsequent fluorescence spectroscopy analysis.Liver tissue samples were wet-digested with nitric acid and stored as acidic digest solutions for analysis by fluorescence spectroscopy. The liver tissue was first freeze dried to minimise analyse loss and to facilitate subsequent sample preparation steps. The tissue was then homogenised to a fine powder by ball-milling in plastic containers. Approximately 0.20-0.25 g of powdered tissue was weighed into a Teflon reaction vessel and 3 ml of HNOFluorescence spectra of liver tissue containing GNPs of different sizes  were obtained using a FluoroMax-2 spectrofluorometer . Fluorescence measurements were made over the wavelength range 250-700 nm using 1 cm path length quartz cuvettes, which were cleaned before each use by sonicating for 5 min in deionised water and then rinsing with deionised water.The 10 and 20 nm GNPs showed spherical morphology with a narrow particle size distribution when dispersed in solution. The mean size for these GNPs was calculated from the TEM images. The mean measured size was 9.45 ± 1.33 nm for 10 nm GNPs and 20.18 ± 1.80 nm for 20 nm GNPs. GNPs of 50 nm diameters, in contrast, were not spherical but hexagonal in TEM images, as shown in Figure The maximum of the surface plasmon band (SPB) of GNPs in solution was shifted from 517 nm to 532 nm when the GNP size changed from 10 nm to 50 nm. This effect was attributed to the surface plasmon oscillation of free electrons. After injecting 20 nm GNPs into the experimental rats, we observed two fluorescence peaks at 438 nm and 487 nm in the digested liver tissue.Figures Figure Figures Figure Figure Figure Nanotechnology has recently emerged as a promising field for the treatment and diagnosis of a variety of diseases . GNPs arThe results of this study indicate that decreasing nanoparticle size, which produces an exponential increase in surface area relative to volume, may make the GNPs more self-reactive  and more prone to interactions with surrounding molecules . Moreover, increased uptake of nanoparticles may lead to accumulation in certain tissues, where the particles may interfere with critical biological functions ,21. We nTo evaluate the time dependence of GNP distribution and aggregation, we administered 50 μl of GNPs of  by daily intraperitoneal injection into rats for periods of 3 or 7 days. At these time points, the bio-distribution of the GNPs was quantitatively measured by fluorescence spectroscopy and TEM. The size of the GNPs strongly influenced the bio-distribution.The very small size of nanoparticles imparts physical and chemical properties that are very different from those of the same material in bulk form. Nanoparticles have a larger surface area to volume ratio compared to bulk materials; they may thus exhibit an enhanced or hindered tendency to aggregate (depending on the surface chemistry), enhanced photoemission, high electrical or heat conductivity, or improved surface catalytic activity ,21.Because nanoparticle surfaces can interact with biological components, nanoparticles may be more reactive than larger particles toward biomolecules. It has been shown, for example, that the severity and the likelihood of inflammatory response transiently increased, within 12 h, following injection of 200 or 100 nm GNPs into experimental animals. GNPs were ultimately trapped by macrophages in the spleen and liver and remained in these tissues until 4 weeks after the single injection .To evaluate the impact of particle size on tissue distribution, we injected rats with 50 μl GNPs of different sizes. The 50 nm GNPs were taken up by liver macrophages faster and to a greater extent than smaller GNPs; the particles were eliminated after uptake. This result is correlated with the inflammatory response of the liver.The present results show that 50 nm GNPs may be cleared via urine and bile rapidly. Nanoparticles for therapeutic use need to have a long retention time in order to encounter and interact with the desired target. However, a long retention time can result in toxic effects in vivo. Thus, route and rate of nanomaterial clearance is an important issue ,29.Absorbed nanoparticles in the systemic circulation can be excreted through various routes, such as the kidneys or bile. Renal clearance of solid nano-sized materials is known to be influenced by particle size and surface charge ,30. In tAll GNPs showed a propensity to accumulate in tissues following injection. The liver tissue distribution of GNPs was size dependent: the smallest particles showed the most widespread organ distribution. Smaller GNPs also showed greater accumulation in cells according to dose-metric treatment. Therefore, it should be taken into consideration that GNPs preferentially target organs with many phagocytic cells, such as kidney, spleen, lung, heart and mesenteric lymph nodes.High electron density and homogeneous shape and size make GNPs highly conspicuous in TEM images. We found that the 10 and 20 nm GNP preparations exhibit spherical morphology, while 50 nm GNPs are hexagonal.The size , shape and concentration of GNPs all contributed to differences in the fluorescence intensities between GNP samples. The time dependence of GNP fluorescence spectra was also dependent on these factors; smaller particles generally exhibited higher fluorescence that increased with GNP infusion time, whereas larger GNPs exhibited lower fluorescence decreasing with infusion time.This study suggests that fluorescence intensity may be a useful diagnostic probe for bioaccumulation and toxicity of gold nanoparticles administered to rats. Moreover, smaller particles, which have an increased ratio of surface area to volume, appear to be more reactive toward one another (resulting in aggregation) and toward biological components in the surrounding environment.The authors declare that they have no competing interests.MAKA and MMM have analyzed the data, interpreted and written the final draft of this manuscript. The animal model used in this study was obtained from the Laboratory Animal Center . MAKA has conceived the study and its design and obtained research grants for this study. Moreover, both authors have read and approved the final manuscript.


Advances in nanotechnology have identified promising candidates for many biological and biomedical applications. Since the properties of nanoparticles (NPs) differ from that of their bulk materials, they are being increasingly exploited for medical uses and other industrial applications. The histological and the histochemical alterations in the renal tissues due to gold nanoparticles (GNPs) have not well documented and have not yet been identified. The aim of the present study was to investigate the particle-size effect of GNPs on the renal tissue in an attempt to address their potential toxicity.A total of 70 healthy male Wistar-Kyoto rats were exposed to GNPs received 50 or 100 μl of GNPs infusion of size  to investigate particle-size effect of GNPs on the renal tissue. Animals were randomly divided into groups, 6 GNPs-treated rats groups and one control group. Groups 1, 2 and 3 received infusion of 50 μl GNPs of size 10 nm (3 or 7 days), size 20 nm (3 or 7 days) and 50 nm (3 or 7 days), respectively; while groups 4, 5 and 6 received infusion of 100 μl GNPs of size 10 nm, size 20 nm and 50 nm, respectively.The histological alterations were mainly seen in the cortex and the proximal renal convoluted tubules were more affected than the distal ones. In comparison with respective control rats, exposure to GNPs doses has produced the following renal tubular alterations: cloudy swelling and renal tubular necrosis. Interstitial alterations included: intertubular blood capillaries dilatation, intertubular hemorrhage and inflammatory cell infiltrations. The glomeruli showed moderate congestion with no hypercelluraity and mesangial proliferation or basement membrane thickening.The induced histological alterations might be an indication of injured renal tubules due to GNPs toxicity that become unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these NPs. These alterations were size-dependent with smaller ones induced more effects and related with time exposure of GNPs. The produced histological alterations may suggest that GNPs interact with proteins and enzymes of the renal tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may induce stress in the renal cells to undergo atrophy and necrosis. More histomorphologcal investigations are needed to address the potential threat of GNPs as a therapeutic and diagnostic tool. Nanoparticles are an intermediate state of matter somewhere between bulk and molecular level. These particles have important application for cell labeling and imaging, drug delivery, biological sensors, diagnostic and therapeutic purposes mainly in cancer and photodynamic therapy -6. StudiGold in its bulk form has long been considered an inert, noble metal with some therapeutic and even medicinal value hence GNPs are thought also to be relatively non-cytotoxic . Yet theAlthough some scientists consider NPs as nontoxic, there are other studies reporting the toxic effects of NPs -16. WhilWhile nanotoxicity research is now gaining attention, little is paid to the effect of size and period of NPs mainly to their distribution and alterations in the tissue ,22. In tA total of 70 healthy male Wistar-Kyoto rats obtained from the Laboratory Animal Center . The rats nearly of the same age (12 weeks old) and weighing 220-240 gm of King Saud University colony were used. Animals were randomly divided into groups, 6 GNPs-treated rats groups and one control group. Following a period of stabilization (7 days), 10, 20 and 50 nm GNPs were administered intraperitonealy at the rate for 3 or 7 days as follows: Group 1: received infusion of 50 μl GNPs of size 10 nm for 3 or 7 days (n = 10); Group 2: received infusion of 50 μl GNPs of size 20 nm for 3 or 7 days (n = 10); Group 3: received infusion of 50 μl GNPs of size 50 nm for 3 or 7 days (n = 10); Group 4: received infusion of 100 μl GNPs of size 10 nm for 3 or 7 days; (n = 10); Group 5: received infusion of 100 μl GNPs of size 20 nm for 3 or 7 days (n = 10); Group 6: received infusion of 100 μl GNPs of size 50 nm for 3 or 7 days; (n = 10); Control group: received no GNPs (n = 10).The rats were maintained on standard laboratory rodent diet pellets and were housed in humidity and temperature-controlled ventilated cages on a 12 h day/night cycle. All experiments were conducted in accordance with the guidelines approved by King Saud University Local Animal Care and Use Committee.Fresh portions of both kidneys from each rat were cut rapidly, fixed in neutral buffered formalin (10%), then dehydrated, with grades of ethanol . Dehydration was then followed by clearing the samples in 2 changes of xylene. Samples were then impregnated with 2 changes of molten paraffin wax, then embedded and blocked out. Paraffin sections (4-5 um) were stained with hematoxylin and eosin  according to Pearse . StainedNo mortality occurred in any of the experimental groups of the present investigation, and no alterations were observed in the appearance and behavior of GNPs treated rats in comparison with the control ones. In comparison with the control group, the following histological alterations were detected in the renal tissue of GNPs treated rats: The following tubular alterations due to GNPs intoxication appeared in the renal tissue of the treated rats.GNPs produced occasional glomerular congestion in the rats exposed to 10 nm or 20 nm particles for 7 days but not in the glomeruli of the rats exposed to 50 nm  s Figure . This dis Figure .The loop of Henle showed very little or no alterations due to GNPs intoxication while occasional cytomegaly and luminal hyaline casts were the only alterations observed in the collecting tubules. The renal tissue of the rats received 50 or 100 μl of 50 nm GNPs for 3 or 7 days showed little or no alterations while none of the above alterations were observed in the renal tissue of any member of the control group Figures . StudiesThe results of the present work showed that the cortex is more affected than medulla due to treatment with GNPs. This could be partly due to uneven distribution of these particles in the tissue of the kidney where about 90% of the total renal blood flow enters the cortex via the bloodstream. Accordingly, a relative high concentration of particles might reach the cortex via the bloodstream than that would enter the medulla.The findings of the present study indicate that the potential toxicity of GNPs is dependent upon the size of these particles. These findings are in line with the reports of Pan et al., 2007  where ceHistological alterations by GNPs exposure as shown in the results of the present work could be an indication of injured renal tissue due to GNPs toxicity that become unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these particles. One might conclude that these alterations are size-dependent with smaller ones induced more damage to renal tissue with relation with the time exposure of GNPs. This might be due to earlier accumulation of the larger NPs in the tissue while the smaller ones stay much longer in the bloodstream due to recirculation.The appearance of renal cells cytoplasmic degeneration and nuclear destruction may suggest that GNPs interact with proteins and enzymes of the renal tissue, interfering with the antioxidant defense mechanism and leading to generation and accumulation of the reactive oxygen species (ROS) which in turn may produce inflammatory response and mitochondrial destruction inducing stress in the renal cells to undergo atrophy, necrosis and programmed cell death.More histomorphologcal, histochemical and ultrastrucural investigations are needed to correlate the biomedical application of GNPs with their therapeutic and diagnostic use in correlation with the size, chemical composition, surface charge, solubility and surface structure of these particles.The authors declare that they have no competing interests.MAKA and BMJ have analyzed data, interpreted and written the final draft of this manuscript. The animal model used in this study was obtained from the Laboratory Animal Center . Dr. MAKA has conceived the study and its design and obtained research grants for this study. Moreover, both authors have read and approved the final manuscript.


Current research focuses on cancer therapy, diagnostics and imaging, although many challenges still need to be solved. However, for the application of gold nanoparticles (GNPs) in therapy and diagnostics it is necessary to know the bioaccumulation and local or systemic toxicity associated to them. The aim of the present study was to investigate the effects of intraperitoneal administration of GNPs on the histological alterations of the heart tissue of rats in an attempt to cover and understand the toxicity and the potential role of GNPs in the therapeutic and diagnostic applications.Animals were randomly divided into 3 GNPs-treated rats groups and one control group (CG). The 10, 20 and 50 nm GNPs were administered intraperitonealy at the rate of 3 or 7 days as follows: Group 1: received infusion of 100 μl GNPs of size 10 nm for 3 or 7 days; Group 2: received infusion of 100 μl GNPs of size 20 nm for 3 or 7 days; Group 3: received infusion of 100 μl GNPs of size 50 nm for 3 or 7 days. Control group: received no GNPs.In comparison with the respective control rats, GNPs-treated rat received 100 μl of 10 and 20 nm particles for 3 days or 7 days demonstrating congested heart muscle with prominent dilated blood vessels, scattered and extravasations of red blood cells, focus of muscle hyalinosis, disturbed muscle fascicles, dense prominent focus of inflammatory cells infiltrate by small lymphocytes and few plasma cells while GNPs-treated rat received 100 μl of 50 nm particles for 3 or 7 days demonstrating benign normal looking heart muscle with normal muscle direction and fascicles, and very few scattered small lymphocytes.The histological alterations induced by intraperitoneal administration of GNPs were size-dependent with smaller ones induced more affects and related with time exposure of GNPs. This study suggests that interaction of GNPs with proteins and various cell types might be evaluated as part of the toxicological assessment in addition to further experiments related to tissues antioxidant enzymes, oxidative parameters, lipid peroxidation, production of free radicals and/or ROS and cytokine, histomorphologcal and ultrastrucural will be performed to cover and understand the toxicity and the potential use of GNPs as therapeutic and diagnostic tool. The NPs are being investigated for gene delivery purposes -3 and caNPs may differ in reactivity and solubility and may interact with all kinds of endogenous proteins, lipids, polysaccharides and cells. A series of tests was proposed for evaluation of the toxicity of NPs used in drug delivery systems . GNPs caGold in its bulk form has been considered an inert, noble metal with some therapeutic and medicinal value. GNPs are thought also to be relatively non-cytotoxic  while thThe use of NPs as drug carriers may reduce the toxicity of the incorporated drug . There aThe particle size-dependent organ distribution of GNPs has been studied in vivo -17. In vIn order to understand and categorize the mechanisms for GNPs toxicity, histological data is needed on the response of living systems to the presence of GNPs of varying size, shape, surface, and exposure duration.The histological and histochemical characterization of the heart tissues due to GNPs has not been documented and identified before. In the present study, an attempt has been made to characterize the possible histological alterations in the heart tissues after intraperitoneal administration of GNPs and, if so, whether are related to the size of these GNPs and the time of exposure.GNPs of different sizes  were purchased. All GNPs used in this study were in aqueous solution at a concentration of 0.01%. The mean size and morphology of these GNPs were evaluated from transmission electron microscope (TEM) images.A total of 40 healthy male Wistar-Kyoto rats were obtained from the Laboratory Animal Center . The rats nearly of the same age (12 weeks old) and weighing 220-240 g of King Saud University colony were used. Animals were randomly divided into 3 GNPs-treated rats groups and one control group (CG). The 10, 20 and 50 nm GNPs were administered intraperitonealy at the rate of 3 or 7 days as follows: Group 1: received infusion of 100 μl GNPs of size 10 nm for 3 or 7 days (n = 10); Group 2: received infusion of 100 μl GNPs of size 20 nm for 3 or 7 days (n = 10); Group 3: received infusion of 100 μl GNPs of size 50 nm for 3 or 7 days (n = 10). Control group: received no GNPs (n = 10).The rats were maintained on standard laboratory rodent diet pellets and housed in humidity and temperature-controlled ventilated cages on a 12 h day/night cycle. All experiments were conducted in accordance with the guidelines approved by King Saud University Local Animal Care and Use Committee.Fresh portions of the heart from each rat were cut rapidly, fixed in neutral buffered formalin (10%), then dehydrated, with grades of ethanol . Dehydration was then followed by clearing the samples in 2 changes of xylene.Samples were then impregnated with 2 changes of molten paraffin wax, then embedded and blocked out. Paraffin sections (4-5 um) were stained with hematoxylin and eosin  according to Pearse . The briThe 10 and 20 nm GNPs show spherical shape while 50 nm GNPs show hexagonal shape. The mean size for GNPs was calculated from the images taken by the transmission electron microscope (TEM): The 10 nm GNPs was of mean size 9.45 ± 1.33 nm, 20 nm GNPs was of mean size 20.18 ± 1.80 and the 50 nm GNPs was of mean size 50.73 ± 3.58 .No mortality occurred for the administration periods 3 and 7 days of GNPs in any of the experimental groups of the present investigation, and no alterations were observed in the appearance and behavior of GNPs treated rats in comparison with the control ones.Control group Figure : MicroscIn comparison with the control group, the following histological alterations were detected in the heart tissue of GNPs-treated rats. These histological alterations were observed in Figures 1) GNPs-treated rat received 100 μl of 10 nm particles for 3 days demonstrating heart muscle with prominent dilated congested blood vessels, scattered and extravasations of red blood cells and few small lymphocytic infiltrate as shown in Figure 22) GNPs-treated rat received 100 μl of 10 nm particles for 7 days demonstrating scattered and extravasations of red blood cells, congested dilated blood vessels, prominent focus of small lymphocytic infiltrate, focus of muscle hyalinosis, disturbed muscle fascicles as shown in Figure 3) GNPs-treated rat received 100 μl of 20 nm particles for 3 days demonstrating dense prominent focus of inflammatory cells infiltrate by small lymphocytes, few plasma cells, prominent congested dilated blood vessels and few scattered extravasations of red blood cells as shown in Figure 4) GNPs-treated rat received 100 μl of 20 nm particles for 7 days demonstrating dense chronic inflammatory cells infiltrate with extravasations of red blood cells as shown in Figure 5) GNPs-treated rat received 100 μl of 50 nm particles for 3 days demonstrating benign normal looking heart muscle with normal muscle direction and fascicles with no pathological effect as shown in Figure 66) GNPs-treated rat received 100 μl of 50 nm particles for 7 days demonstrating benign normal looking heart muscle, very few scattered small lymphocytes as shown in Figure The histological heart alterations induced by intraperitoneal administration of GNPs were size-dependent with smaller ones induced more affects and related with time exposure of GNPs.This infiltration of GNPs was more prominent after 7 days of administration and in rats received 10 and 20 nm GNPs than those received 50 nm GNPs. The histological heart alterations may suggest that GNPs could interfere with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may imitate an inflammatory response. Inflammatory cells infiltration was seen in the portal triads and the perioral zones of GNPs treated rats. The infiltrate cells were mainly lymphocytes and plasma cells .GNPs were more strongly oxidizing as evidenced by lipid peroxidation ,25 as weIt has been reported that 5 nm GNPs caused significantly greater oxidative stress and cytotoxicity effects than larger ones -29. The Several possible mechanisms of action for the toxicity of particles including injury of epithelial tissue , inflammNPs are nearly of same dimensions as some biological molecules such as proteins and nucleic acids. Many of these biomolecules consist of long macromolecular chains which are folded and shaped by cooperative and weak interaction between side groups. The GNPs may intrude into these complex folded structures.GNPs activate the phagocytic activity of the sinusoidal cells by increasing the number of Kupffte cells to help in removing the accumulated GNPs where lysosomes are involved in the intracellular breakdown into small metabolic products. The produced Kupffer cells hyperplasia might be correlated with the amount of injurious to the hepatic tissue induced by GNPs intoxication and represents a defense mechanism of detoxification. Kupffer cell hyperplasia is contributed to hepatic oxidative stress -23,38.+ due to GNPS effects. Cellular swelling might be accompanied by leakage of lysosomal hydrolytic enzymes that lead to cytoplasmic degeneration and macromolecular crowding [This scattered cytoplasmic vacuolization might indicate toxicity effect that exhibited as a result of disturbances of membranes function which leads to massive influx of water and Nacrowding .Fatty change was observed in some swelling hepatocytes of rats exposed to 100 μl of 10 nm GNPs and to lesser extent in the ones exposed to larger particles. This hepatic liposis was more prominent in rat exposed to GNPs for 7 days than those received the treatment for 3 days -23. HepaThe rats received 10 and 20 nm GNPs showed hemorrhage and excess extravasation of red blood cells. Less disruption was observed in rats exposed to 50 nm GNPs while more damage was detected after 7 days than 3 days of GNPs exposure. This alteration might indicate heart muscle damage and congestion by GNPs exposure.None of the above alterations were observed in the heart muscle of any member of the control group.The interaction of NPs with living systems is also affected by the characteristic dimensions. As noted above, GNPs, of smaller size, may reach inside biomolecules, a situation not possible for larger GNPs. It has been reported that inhaled NPs reach the blood and may reach other target sites such as the liver, heart or blood cells ,41.Reduction in size results in an enormous increase of surface to volume ratio, so relatively more molecules of the chemical are present on the surface, thus enhancing the intrinsic toxicity. This may be one of the reasons that smaller GNPs are generally more toxic than larger particles of the same insoluble material when compared on a mass dose base .In the present study we have not measured GNPs concentration in urine and feces, but this point will be taken into our consideration in other new additional experiments.Further experiments related to tissues antioxidant enzymes, oxidative parameters, lipid peroxidation, production of free radicals and/or ROS and cytokine, histomorphologcal and ultrastrucural will be performed to cover and understand the toxicity and the potential use of GNPs as therapeutic and diagnostic tool.In comparison with the respective control rats, histological alterations induced in the heart tissue exposure could be an indication of congested heart muscle with dilated blood vessels, extravasations of red blood cells, muscle hyalinosis, disturbed muscle fascicles, inflammatory cells infiltrate by small lymphocytes and plasma cells due to GNPs toxicity that became unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these particles. One might conclude that these alterations are size-dependent with smaller ones induced more damage with relation to the time exposure of GNPs.The GNPs-treated rat received 100 μl of 50 nm particles for 3 or 7 days demonstrating benign normal looking heart muscle with normal muscle direction and fascicles, very few scattered small lymphocytes, and with no other pathological effects.One mechanism of toxicity of NPs is likely to be induction of ROS and the consequential oxidative stress in cells and organs. The appearance of congested heart muscle with prominent dilated blood vessels and focus of inflammatory cells infiltrate by small lymphocytes and plasma cells may suggest that GNPs interact with proteins and enzymes of the hepatic tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may induce stress.The author declares that he has no competing interests.AMAK has analyzed data, interpreted and written the final draft of this manuscript. The animal model used in this study was obtained from the Laboratory Animal Center . AMAK has conceived the study and its design and obtained research grants for this study. The authors have read and approved the final manuscript.


Gold nanoparticles (GNPs) have important application for cell labeling and imaging, drug delivery, diagnostic and therapeutic purposes mainly in cancer. Nanoparticles (NPs) are being increasingly exploited for medical applications. The aim of the present study was to investigate the particle-size and period effects of administration of GNPs on the renal tissue in an attempt to address their potential toxicity.A total of 70 healthy male Wistar-Kyoto rats were exposed to GNPs received 50 or 100 μl of GNPs infusion of size  to investigate particle-size effect of GNPs on the renal tissue. Animals were randomly divided into groups, 6 GNPs-treated rats groups and one control group. Groups 1, 2 and 3 received infusion of 50 μl GNPs of size 10 nm (3 or 7 days), size 20 nm (3 or 7 days) and 50 nm (3 or 7 days), respectively; while groups 4, 5 and 6 received infusion of 100 μl GNPs of size 10 nm, size 20 nm and 50 nm, respectively. Stained sections of control and treated rats kidneys were examined for renal tissue alterations induced by GNPs.In comparison with respective control rats, exposure to GNPs doses has produced the following renal tubular alterations: cloudy swelling, vacuolar degeneration, hyaline droplets and casts, anisokaryosis, karopyknosis, karyorrhexis and karyolysis. The glomeruli showed moderate congestion with no hypercelluraity, mesangial proliferation or basement membrane thickening. The histological alterations were mainly seen in the cortex and the proximal renal convoluted tubules were more affected than the distal ones.The induced histological alterations might be an indication of injured renal tubules due to GNPs toxicity that became unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these NPs. The findings may suggest that GNPs interact with proteins and enzymes of the renal tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may induce stress in the renal cells to undergo atrophy and necrosis. The produced alterations were size-dependent with smaller ones induced more affects and related with time exposure of GNPs. Nanoparticles are an intermediate state of matter somewhere between bulk and molecular level. These particles have important application for cell labeling and imaging, drug delivery, biological sensors, diagnostic and therapeutic purposes mainly in cancer and photodynamic therapy -6. StudiGold in its bulk form has long been considered an inert, noble metal with some therapeutic and even medicinal value hence GNPs are thought also to be relatively non-cytotoxic . Yet theAlthough some scientists consider NPs as nontoxic, there are other studies reporting the toxic effects of NPs -16. WhilWhile nanotoxicity research is now gaining attention, little is paid to the effect of GNPs size mainly to their distribution and alterations in the tissue ,22. The A total of 70 healthy male Wistar-Kyoto rats obtained from the Laboratory Animal Center . The rats nearly of the same age (12 weeks old) and weighing 220-240 gm of King Saud University colony were used. Animals were randomly divided into groups, 6 GNPs-treated rats groups and one control group. Following a period of stabilization (7 days), 10, 20 and 50 nm GNPs were administered intraperitonealy at the rate for 3 or 7 days as follows: Group 1: received infusion of 50 μl GNPs of size 10 nm for 3 or 7 days (n = 10); Group 2: received infusion of 50 μl GNPs of size 20 nm for 3 or 7 days (n = 10); Group 3: received infusion of 50 μl GNPs of size 50 nm for 3 or 7 days (n = 10); Group 4: received infusion of 100 μl GNPs of size 10 nm for 3 or 7 days; (n = 10); Group 5: received infusion of 100 μl GNPs of size 20 nm for 3 or 7 days (n = 10); Group 6: received infusion of 100 μl GNPs of size 50 nm for 3 or 7 days; (n = 10); Control group: received no gold nanoparticles (n = 10).The rats were maintained on standard laboratory rodent diet pellets and were housed in humidity and temperature-controlled ventilated cages on a 12 h day/night cycle. Two animals from each group were killed by dislocation of the neck at intervals of 3 and 7 days of treatment with GNPs. All experiments were conducted in accordance with the guidelines approved by King Saud University Local Animal Care and Use Committee.Fresh portions of both kidneys from each rat were cut rapidly, fixed in neutral buffered formalin (10%), then dehydrated, with grades of ethanol . Dehydration was then followed by clearing the samples in 2 changes of xylene. Samples were then impregnated with 2 changes of molten paraffin wax, then embedded and blocked out. Paraffin sections (4-5 um) were stained with hematoxylin and eosin stain according to Pearse . StainedNo mortality occurred in any of the experimental groups of the present investigation, and no alterations were observed in the appearance and behavior of GNPs treated rats in comparison with the control ones. In comparison with the control group, the following histological alterations were detected in the renal tissue of GNPs treated rats:. No hypercellularity, mesangial proliferation or glomerular basement membranes thickening were detected in the glomeruli of all GNPs treated rats. Occasional dilatation of glomerular tuft blood capillaries was observed. These little alteration showed by the glomeruli might be due to the glomerular basement membrane which forms a barrier that prevents nanoparticles accumulation. Terentyuk et al., 2009 [GNPs produced occasional glomerular congestion in the rats exposed to 10 nm or 20 nm particles for 7 days but not in the glomeruli of the rats exposed to 50 nm l., 2009  reportedThe following tubular alterations due to GNPs intoxication appeared in the renal tissue of the treated rats.Cloudy swelling: renal tubules epithelial lining exhibited cloudy swelling with pale cytoplasm and poorly delineated and displaced nuclei in all GNPs treated rats. This alteration was more prominent in the proximal convoluted tubules than the distal ones and with more swelling with 100 μl dose than 50 μl one and with 10 nm and 20 nm size particles than the larger ones . Cytoplasmic swelling might be exhibited as a result of disturbances of membranes function that lead to massive influx of water and Na+ due to GNPS effects. This alteration might be accompanied by leakage of liposome hydrolytic enzymes that lead to cytoplasmic degeneration and macromolecular crowding [crowding .Vacuolar degeneration: vacuolization of the renal cells was seen and increased in severity in the renal tubules of rats received 100 μl of 10 or 20 nm GNPs with less or no vacuolar degeneration with 50 nm particles. More vacuolar degeneration was observed in the renal cells of rats exposed to 7 days than ones exposed to 3 days . Droplets appearance is associated with protein metabolism disturbances. This alteration was not seen in the renal tissue of rats exposed to 50 nm particles. Hyaline casts were also seen in the lumen of some distal convoluted renal tubules .s Figure . Droplets Figure .Anisokaryosis: variable nuclei sizes were observed in some renal cells. This change became apparent after 7 days of 50 nm GNPs administration . Some studies indicate that nuclear polymorphism is seen in dysplasia and carcinomatous lesion [s lesion .Nuclear pyknosis: sections of GNPs-treated kidneys developed pyknosis in some epithelium lining cells of the proximal tubules with lesser extent in the distal ones. This alteration was seen in all GNPs-treated rats with 10 and 20 nm particles . Pyknotic nuclei exhibited clumping and condensation of the chromatin materials in the periphery of the nuclei together with irregularity nuclear membranes. Karyopyknosis is an irreversible condensation of chromatin in the nucleus of a cell undergoing necrosis or apoptosis [s Figure . Pyknotipoptosis .Karyorrhexis: some renal cells of rats received 10 and 20 nm GNPs showed nucleoli disappearance . This nuclear damage was more prominent aftere Figure . This nu7 days of exposure to NPs. Karyorrhexis is a sort of destructive fragmentation of the nucleus proceeded by pyknosis and is followed by karyolysis .Karyolysis: this alteration appeared mainly in the kidney of GNPs-treated rats exposed to 100 μl of 20 nm size particles  which in turn may inflammatory response and mitochondrial destruction inducing stress in the renal cells to undergo atrophy and necrosis and programmed cell death.More histomorphologcal, histochemical and ultrastrucural investigations are needed to correlate the biomedical application of GNPs with the potential threat of their therapeutic and diagnostic use in correlation with the size, chemical composition, surface charge, solubility and surface structure of these particles.The authors declare that they have no competing interests.MAKA and BMJ have analyzed data, interpreted and written the final draft of this manuscript. The animal model used in this study was obtained from the Laboratory Animal Center . MAKA has conceived the study and its design and obtained research grants for this study. Moreover, both authors have read and approved the final manuscript.


Nanoparticles (NPs) can potentially cause adverse effects on organ, tissue, cellular, subcellular and protein levels due to their unusual physicochemical properties. Advances in nanotechnology have identified promising candidates for many biological and biomedical applications. The aim of the present study was to investigate the particle-size, dose and exposure duration effects of gold nanoparticles (GNPs) on the hepatic tissue in an attempt to cover and understand the toxicity and their potential therapeutic and diagnostic use.A total of 70 healthy male Wistar-Kyoto rats were exposed to GNPs received 50 or 100 ul of GNPs infusion of size  to investigate particle-size, dose and exposure duration effects of GNPs on the hepatic tissue.In comparison with respective control rats, exposure to GNPs doses has produced alterations in the hepatocytes, portal triads and the sinusoids. The alterations in the hepatocytes were mainly vacuolar to hydropic degeneration, cytopasmic hyaline vacuolation, polymorphism, binucleation, karyopyknosis, karyolysis, karyorrhexis and necrosis.+ due to GNPs effects accompanied by leakage of lysosomal hydrolytic enzymes that lead to cytoplasmic degeneration and macromolecular crowding. Hydropic degeneration is a result of ion and fluid homestasis that lead to an increase of intracellular water. The vacuolated swelling of the cytoplasm of the hepatocytes of the GNPs treated rats might indicate acute and subacute liver injury induced by the GNPs. Binucleation represents a consequence of cell injury and is a sort of chromosomes hyperplasia which is usually seen in regenerating cells. The induced histological alterations might be an indication of injured hepatocytes due to GNPs toxicity that became unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these NPs. These alterations were size-dependent with smaller ones induced the most effects and related with time exposure of GNPs. The appearance of hepatocytes cytoplasmic degeneration and nuclear destruction may suggest that GNPs interact with proteins and enzymes of the hepatic tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may induce stress in the hepatocytes to undergo atrophy and necrosis. More histomorphologcal, histochemical and ultrastrucural investigations are needed in relation of the application of GNPs with their potential role as a therapeutic and diagnostic tool.The hepatocytes swelling might be exhibited as a result of disturbances of membranes function that lead to massive influx of water and Na The rats exposed to aerosols of GNPs revealed that the NPs were rapidly taken into the system with the highest accumulation in the lungs, aorta, esophagus and olfactory bulb . MoreoveAlthough some scientists consider NPs as nontoxic, there are other studies reporting the toxic effects of NPs -5. AlthoGold in its bulk form has long been considered an inert, noble metal with some therapeutic and even medicinal value hence GNPs are thought also to be relatively non-cytotoxic . Yet theThe histological and the histochemical characterization in the hepatic tissues due to GNPs are not documented and have not yet been identified. In the present study, an attempt has been made to characterize the possible histological alterations in the hepatic tissues following experimental GNPs and, if so, whether are related to the size of these NPs and the time of exposure.The present study was carried out to investigate the particle-size, dose and exposure duration of GNPs on the hepatic tissue in an attempt to cover and understand the toxicity and their potential therapeutic and diagnostic use in relation with the time of exposure.A total of 70 healthy male Wistar-Kyoto rats obtained from the Laboratory Animal Center . The rats nearly of the same age (12 weeks old) and weighing 220-240 gm of King Saud University colony were used. Animals were randomly divided into groups, 12 GNPs-treated rats groups and one control group (NG). Following a period of stabilization (7 days), 10, 20 and 50 nm GNPs were administered intraperitonealy at the rate for 3 or 7 days as follows: Group 1: received infusion of 50 μl GNPs of size 10 nm for 3 or 7 days (n = 10); Group 2: received infusion of 50 μl GNPs of size 20 nm for 3 or 7 days (n = 10); Group 3: received infusion of 50 μl GNPs of size 50 nm for 3 or 7 days (n = 10); Group 4: received infusion of 100 μl GNPs of size 10 nm for 3 or 7 days; (n = 10); Group 5: received infusion of 100 μl GNPs of size 20 nm for 3 or 7 days (n = 10); Group 6: received infusion of 100 μl GNPs of size 50 nm for 3 or 7 days; (n = 10); Control group: received no gold nanoparticles (n = 10).The rats were maintained on standard laboratory rodent diet pellets and were housed in humidity and temperature-controlled ventilated cages on a 12 h day/night cycle. Two animals from each group were killed by dislocation of the neck at intervals of 3 and 7 days of treatment with GNPs. All experiments were conducted in accordance with the guidelines approved by King Saud University Local Animal Care and Use Committee.Fresh portions of the lateral lobes of the liver from each rat were cut rapidly, fixed in neutral buffered formalin (10%), then dehydrated, with grades of ethanol . Dehydration was then followed by clearing the samples in 2 changes of xylene. Samples were then impregnated with 2 changes of molten paraffin wax, then embedded and blocked out. Paraffin sections (4-5 um) were stained with hematoxylin and eosin the conventional histological and stain according to Pearse . StainedThe 10 and 20 nm GNPs show spherical shape while the 50 nm GNPs show hexagonal shape. The mean size for GNPs was calculated from the images taken by the transmission electron microscope (TEM). The 10 nm GNPs was of mean size 9.45 ± 1.33 nm, 20 nm GNPs was of mean size 20.18 ± 1.80 and the 50 nm GNPs was of mean size 50.73 ± 3.58 .GNPs-normal rat demonstrating normal hepatocytes are shown in Figure + due to GNPS effects. Cellular swelling might be accompanied by leakage of lysosomal hydrolytic enzymes that lead to cytoplasmic degeneration and macromolecular crowding [hepatocytes exhibited cloudy swelling with pale cytoplasm and poorly delineated and displaced nuclei in all GNPs treated rats. This ballooning degeneration was more prominent with 100 μl dose than 50 μl one and with 10 nm size particles than the larger ones Figure . This swcrowding .vacuolization of the hepatocytes cytoplasm was seen and increased in severity in the liver of rats received 100 μl of 10 nm GNPs with less vacuolar degeneration with larger ones. More vacuolar degeneration was observed in the hepatocytes of rats exposed to 7 days than ones exposed to 3 days Figure . Hydropiinclusions similar to Mallory hyaline bodies and hayaline vacuolations were detected in the cytoplasm of some hepatocytes of rats received 100 μl of 10 nm GNPs. This alteration was less prominent in rats exposed to larger particles Figures  and 2d.variable nuclei sizes were observed in some hepatocytes. This change became apparent after 7 days of 50 nm GNPs administration. Some studies indicate that nuclear polymorphism is seen in hepatic dysplasia and carcinomatous lesion .pyknotic nuclei were seen in some hepatocytes of GNPs treated rats. Some pyknotic hepatocytes of rats received 100 μl of 50 nm size particles exhibited clumping and condensation of the chromatin materials in the periphery of the nuclei together with irregularity nuclear membranes Figures  and 3b. some hepatocytes of rats received 50 nm GNPs showed nucleoli disappearance Figure . This nuthis alteration appeared mainly in the liver of GNPS-treated rats exposed to 100 ul of 50 nm size particles Figure . Karyolyoccasional binucleation and to lesser extent polynucleation were observed in GNPs treated rats. This change was more prominent in rats exposed to 100 μl of 50 nm size GNPs Figures  and 4b. sporadic spotty well-defined necrosis was noticed in some hepatocytes of GNPs treated rats Figure . The inshepatic sinusoids were more dilated in rats received 50 μl of 10 nm GNPs than those exposed to larger particles Figure . This alAbdelhalim and Bashir, 2011 have reported that GNPs-treated rat received 100 μl of 10 nm particles for 7 days demonstrating apoptotic characterization, GNPs-treated rat received 100 μl of 10 nm particles for 3 days demonstrating inflammatory cell infiltration, GNPs-treated rat received 50 μl of 10 nm particles for 7 days demonstrating Kupffer cells hyperplasia, GNPs-treated rat received 100 μl of 10 nm particles for 7 days demonstrating hepatic fatty degeneration and GNPs-treated rat received 50 μl of 20 nm particles for 7 days demonstrating hepatic central vein intima disruption .None of the above alterations were observed in the liver of any member of the control group Figure .Histological alterations by GNPs exposure as shown in the results of the present work could be an indication of injured hepatocytes due to GNPs toxicity that become unable to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these particles. One might conclude that these alterations are size-dependent with smaller ones induced more damage with relation with the time exposure of GNPs.The appearance of hepatocytes cytoplasmic degeneration and nuclear destruction may suggest that GNPs interact with proteins and enzymes of the hepatic tissue interfering with the antioxidant defense mechanism and leading to reactive oxygen species (ROS) generation which in turn may induce stress in the hepatocytes to undergo atrophy and necrosis.More histomorphological, histochemical and ultrastructural investigations are needed to correlate the biomedical application of GNPs with the potential threat of their therapeutic and diagnostic use.The authors declare that they have no competing interests.MAKA and BMJ have analyzed data, interpreted and written the final draft of this manuscript. The animal model used in this study was obtained from the Laboratory Animal Center . MAKA has conceived the study and its design and obtained research grants for this study. Moreover, both authors have read and approved the final manuscript.


The process of microstructure evolution during sintering was clearly distinguished from 2D and 3D reconstructed images. Typical sintering parameters such as sintering neck size, porosity, and particle size of the sample were presented for quantitative analysis of the influence on the mechanical properties and the sintering kinetics during microwave sintering. The neck size-time curve was obtained and the neck growth exponent was 7.3, which indicated that surface diffusion was the main diffusion mechanism; the reason was the eddy current loss induced by the external microwave fields providing an additional driving force for mass diffusion on the particle surface. From the reconstructed images and the curve of porosity and average particle size versus temperature, it was believed that the presence of liquid phase aluminum accelerated the densification and particle growth.In order to study the influence on the mechanical properties caused by microstructure evolution of metal powder in extreme environment, 3D real-time observation of the microstructure evolution of Al-Ti mixed powder in high temperature and microwave compound fields was realized by using synchrotron radiation computerized topography (SR-CT) technique; the spatial resolution was enhanced to 0.37  A lot of the metals and alloys with excellent properties are prepared by powder metallurgy at high temperature , 2. MicrSynchrotron radiation X-ray computed tomography (SR-CT) technique is such a powerful imaging tool developed in recent years , 7. It hμm/pixel. Phase recovery method was used to get high-contrast qualitative microtomography, and microstructure evolution during sintering was clearly distinguished from 2D and 3D reconstructed images, such as sintering neck size, porosity, and particle size of the sample. The size of the sintering neck in each cross section image was calculated by using watershed algorithm, and typical sintering parameters during sintering were presented for quantitative analysis of the influence on the mechanical properties and the sintering kinetics during microwave sintering.In this paper, 3D microstructure characterization of Ti-Al metal powder mixture in high temperature and microwave compound fields was studied in situ, and the spatial resolution was firstly increased to 0.37 The SR-CT technique is a testing method by which the specimen passed through by synchrotron radiation. X-ray is placed on a rotation device and the projection images of the specimen are received by an X-ray charge-coupled device (CCD). One projection image is collected each time when the specimen turns for an angle. After obtaining a set of projection data, reconstruction algorithm is used to obtain the internal microstructure of the sectional images. The 3D images of the microstructure can be obtained from a series of sectional images.μm. The microwave sintering experiment was carried out on the BL13W1 beamline at Shanghai Synchrotron Radiation Facility ; the energy of the beam ranged from 8 to 72 KeV. Considering the X-ray absorption coefficient of Al and Ti, an X-ray with 26 KeV was applied.In the experiment, Al and Ti were selected because they were more commonly used in high-performance metals and alloys. The elemental Al and Ti powders were 300~600 mesh and mixed in the ball mill with alcohol for 10 hours to prepare 87Al-13Ti (wt.%) and then dried in a vacuum oven. The mixed alloy powder was filled into a quartz tube with an inner diameter of 260 A set of unique equipment specifically for the 3D imaging of samples using synchrotron of X-ray had been designed ; the powDue to the sampling capacity limit of CCD target surface, size of the view field would decrease with the improvement of the resolution; therefore, precise positioning of the sample was critical to achieve high resolution SR-CT experiment. The relationship between field of view size and resolution is shown in μm/pixel successfully.In order to carry out high resolution experiments, we developed a special multidimensional high-precision translation and rotation equipment; the design is shown in When there is a certain distance between CCD and the sample, phenomenon of absorption and phase shift occur simultaneously, the information received by CCD includes both absorption and phase information, and the phase contrast image cannot directly reflect the phase and structural information of the sample. X-ray propagation-based phase contrast CT (PPCT) method , 11 was The exposure time would be longer and more projectors would be required in high resolution SR-CT experiment, which led to significant changes in microstructure in the same group of projections, and because the intensity of X-ray decreased with time, the background intensity of projections would be inconsistent. Therefore, in order to increase data acquisition speed, the number of projections would be reduced. Using the inverse projection method could acquire high-quality reconstructed images with fewer projections. By using this method, a series of cross-sectional images at different sintering times was produced, from which 3D images were obtained by applying a 3D rendering algorithm. The 3D reconstructed images of the same microstructure at different sintering times are shown in Sintering necks which initiate and grow during sintering connect loose powders to form a solid metal material. The tensile strength of the metals can be significantly improved by improving the sintering neck structure. Neck growth is a basic phenomenon of sintering, and it plays an important role in determining the main diffusion mechanism and calculating the diffusion coefficient of the material . Therefox and a represent sintering neck and particle size, respectively, t is the sintering time, and different n represents different main diffusion mechanisms. The fourth state is not taken into account because of the presence of liquid aluminum. From x) and log(t), and the slope is 0.1361; the value of inverse slope equals 7.3, indicating that surface diffusion was the dominant diffusion mechanism, which was caused by conductance characteristic of metals. Al and Ti are electrical conductors; the eddy current loss induced by the external microwave fields constitutes the main loss mechanism. This eddy current is along the particle surface layer with the thickness about 1 μm and will probably provide an additional driving force for mass diffusion on the particle surface. Therefore, on the Al and Ti particle surface, the mass transformation process was promoted preferentially.The dynamics of stable neck growth as summarized by Kuczynski is shown in the formula below :(1)(xa)Pores of a material reduce its mechanical properties, such as compressive strength, modulus, shear strength, and flexural strength . With thμm/pixel during microwave sintering, which demonstrated that high-resolution experiments under electromagnetic and high temperature compound fields were feasible. The reconstructed 3D images provided good understanding of various sintering phenomena. Parameters that typically influenced the mechanical properties such as neck size, porosity, and average particle size were analyzed. The neck size-time curve was obtained and the neck growth exponent was 7.3, which indicated that surface diffusion was the main diffusion mechanism, and the reason was the eddy current loss induced by the external microwave fields providing an additional driving force for mass diffusion on the particle surface. From the reconstructed images and the curve of average particle size versus temperature, it was believed that the presence of liquid phase aluminum accelerated the particle growth and densification. In this exploratory study, only a small part of the data acquired by SR-CT was used for quantitative statistical analysis. The 3D reconstructed images represent the complete evolution of microstructure in high temperature and microwave compound fields, such as the smoothness and relative positions of particles, the volume shrinkage of the powder system, and collapse caused by thermal stress; all of these parameters are vital and need to be studied, and they contain the kinetic and thermodynamic information which directly determine the performance of the materials. The challenge is to decouple the information and find the key influence parameters used to calculation model, which makes the study on the sintering more thorough.Based on the SR-CT technique, microstructure evolution of Ti-Al metal powder mixture was observed, and through the design of equipment and the introduction of excellent reconstruction method, the spatial resolution was firstly increased to 0.37