Bioaccumulation of microplastics in decedent human brains

Abstract

Key Aspects

• MNP Presence in Human Tissues: The study's primary finding is the confirmation of micro- and nanoplastic (MNP) presence in human kidney, liver, and brain tissues. This was achieved through a combination of analytical techniques. The presence of MNPs in the brain, a previously unexplored area, is particularly significant, raising concerns about potential neurological impacts.
• Methodological Approach: The research utilized a multi-pronged methodological approach, employing pyrolysis gas chromatography–mass spectrometry, attenuated total reflectance–Fourier transform infrared spectroscopy, and electron microscopy with energy-dispersive spectroscopy. These complementary techniques provide a robust and comprehensive analysis of MNPs, addressing the limitations of individual methods in detecting particles of various sizes and compositions. This strengthens the validity and reliability of the findings.
• Tissue-Specific MNP Characteristics: The study found that brain tissues contained a higher proportion of polyethylene compared to liver or kidney tissues. Additionally, electron microscopy revealed that the MNPs in the brain were primarily nanoscale, shard-like fragments. These findings suggest tissue-specific differences in MNP accumulation and morphology, which could have implications for understanding the mechanisms of MNP uptake and distribution.
• Temporal Increase in MNP Concentration: The research revealed a significant increase in MNP concentrations in both liver and brain samples over time (comparing samples from 2016 and 2024). This finding supports the notion of increasing human exposure to MNPs, paralleling the rising global environmental concentrations. This temporal trend highlights the growing urgency of understanding the long-term health consequences of MNP exposure.
• MNP Accumulation in Dementia Cases: A key finding was the observation of greater MNP accumulation in the brains of individuals with a documented dementia diagnosis, particularly in cerebrovascular walls and immune cells. While this association doesn't prove causation, it raises important questions about a potential link between MNP exposure and neurodegenerative diseases. This warrants further investigation into the potential role of MNPs in dementia pathogenesis.

Strengths

• Clear Statement of Main Finding

  The abstract clearly presents the central finding: the detection and quantification of MNPs in human tissues, particularly the brain.

  "Complementary methods for the robust detection of tissue MNPs...confirm the presence of MNPs in human kidney, liver and brain." (Page 1)
• Multi-methodological Approach

  The abstract succinctly describes the multi-methodological approach used, enhancing the credibility of the findings.

  "Complementary methods for the robust detection of tissue MNPs, including pyrolysis g as c hr om at og raphy–mass spectrometry, attenuated total reflectance–Fourier transform infrared spectroscopy and electron microscopy with energy-dispersive spectroscopy..." (Page 1)
• Concise Summary of Results

  It concisely summarizes the key results, including differences in MNP composition and concentration across tissues and over time.

  "Brain tissues harbor higher proportions of polyethylene compared to the composition of the plastics in liver or kidney...with increasing MNP concentrations over time in both liver and brain samples (P = 0.01)." (Page 1)
• Highlighting of Novel Finding

  The abstract highlights a novel and significant finding regarding MNP accumulation in dementia cases.

  "Finally, even greater accumulation of MNPs was observed in a cohort of decedent brains with documented dementia diagnosis..." (Page 1)
• Emphasis on Broader Implications

  The abstract appropriately concludes by emphasizing the broader implications and need for further research.

  "These results highlight a critical need to better understand the routes of exposure, uptake and clearance pathways and potential health consequences of plastics in human tissues, particularly in the brain." (Page 1)

Suggestions for Improvement

• Explicitly State Novelty

  This high-impact improvement is crucial for placing the study in the context of existing literature and clarifying its novelty. The abstract, as the initial point of contact for most readers, should immediately establish the study's unique contribution.

  "Complementary methods for the robust detection of tissue MNPs...confirm the presence of MNPs in human kidney, liver and brain." (Page 1)

  Implementation: Add a sentence explicitly stating the novelty of the research, such as: "To our knowledge, this is the first study to quantify micro- and nanoplastics in human brain tissue."

• Justify Multi-method Approach

  This medium-impact change would improve the clarity and completeness of the abstract. While the abstract mentions "complementary methods," it doesn't specify *why* this multi-method approach is crucial. Briefly explaining the limitations of individual methods and the need for a combined approach strengthens the rationale.

  "Complementary methods for the robust detection of tissue MNPs, including pyrolysis g as c hr om at og raphy–mass spectrometry, attenuated total reflectance–Fourier transform infrared spectroscopy and electron microscopy with energy-dispersive spectroscopy..." (Page 1)

  Implementation: Add a phrase explaining the need for complementary methods, such as: "...due to the limitations of individual techniques in detecting and characterizing MNPs across a range of sizes and compositions."

• Specify Dementia Types

  This medium-impact change would enhance the abstract's informativeness. While "dementia diagnosis" is mentioned, specifying the *types* of dementia observed would provide valuable context, given the heterogeneity of dementia conditions.

  "Finally, even greater accumulation of MNPs was observed in a cohort of decedent brains with documented dementia diagnosis..." (Page 1)

  Implementation: Modify the sentence to include the types of dementia, such as: "...in a cohort of decedent brains with documented dementia diagnoses, including Alzheimer's disease and vascular dementia..."

• Include MNP Size Range

  This low-impact improvement would add a crucial quantitative element to the abstract. Including the range of MNP sizes detected would provide valuable context for understanding the scope of the findings.

  "Environmental concentrations of anthropogenic microplastic and nanoplastic (MNP), polymer-based particulates ranging from 500 µm in diameter down to 1 nm..." (Page 1)

  Implementation: Add a phrase specifying the size range, such as: "...ranging from 500 µm in diameter down to 1 nm, have increased exponentially..." or "...which present largely as nanoscale shard-like fragments."

• Clarify Time Comparison

  This low-impact improvement would provide context for the temporal findings. Specifying the years compared (2016 vs. 2024) would make the abstract more self-contained.

  "the time of death (2016 versus 2024) was a significant factor, with increasing MNP concentrations over time..." (Page 1)

  Implementation: Change "the time of death (2016 versus 2024)" to "samples collected in 2016 versus 2024".

Introduction

Key Aspects

• Rising Environmental MNP Concentrations: The introduction establishes the growing concern over environmental micro- and nanoplastics (MNPs), which are polymer-based particles ranging from 500 μm to 1 nm. These particles have been increasing exponentially in the environment over the last half-century, raising concerns about human exposure and potential health effects. The pervasive nature of MNPs makes understanding their impact on human health a critical area of research.
• Uncertainty of MNP Toxicity in Humans: The introduction highlights the uncertainty surrounding the extent of harm caused by MNPs to humans. While some studies link MNP presence to increased inflammation and adverse cardiovascular events, and controlled studies show MNPs can exacerbate disease, the relevance of these findings to actual human exposures remains unclear. This uncertainty underscores the need for research focused on human tissue distribution and internal MNP dosage.
• Knowledge Gap: Tissue Distribution and Internal Dose: The introduction identifies a critical knowledge gap: the lack of understanding regarding the tissue distribution and internal dose of MNPs in humans. This gap hinders the interpretation of controlled exposure studies, as the relationship between exposure levels and actual tissue concentrations is not well-defined. Addressing this gap is crucial for accurately assessing the risks associated with MNP exposure.
• Limitations of Existing Detection Methods: The introduction summarizes existing research, noting that visual microscopic spectroscopy methods have identified MNPs in organs like the lungs, intestines, and placenta. However, these methods are often limited to larger particles (>5 μm), excluding smaller nanoplastics. This limitation highlights the need for more comprehensive detection methods, such as the Py-GC/MS approach used in this study.
• Importance of Internal Dose Assessment: The introduction sets the stage for the current study by referencing the toxicological principle that 'dose makes the poison'. It emphasizes that understanding the internal dose of MNPs within human tissues is essential for interpreting existing research and accurately assessing risk. This study seeks to address this critical need by quantifying MNPs in various human organs.

Strengths

• Effective Contextualization

  The introduction effectively establishes the context by highlighting the increasing environmental concentrations of microplastics and nanoplastics (MNPs) and the associated concerns about human exposure and health.

  "Rising global concentrations of environmental microplastics and nanoplastics (MNPs) drive concerns for human exposure and health outcomes." (Page 1)
• Clear Identification of Knowledge Gap

  The introduction clearly states the knowledge gap regarding the tissue distribution and internal dose of MNPs in humans, which is crucial for interpreting controlled exposure studies.

  "what is not clearly understood is the tissue distribution and internal dose of MNPs in humans, which confounds our ability to interpret the controlled exposure study results." (Page 1)
• Summary of Previous Research and Limitations

  It succinctly summarizes previous research on MNP detection in organs, while also pointing out the limitations of existing methods.

  "So far, visual microscopic spectroscopy methods have identified particulates in organs, such as the lungs, intestine7 and placenta8. These methods are often limited to larger (>5 µm) particulates; thus, smaller nanoplastics are unintentionally excluded." (Page 1)

Suggestions for Improvement

• Explicitly State Novelty

  This high-impact improvement is essential to immediately establish the study's unique contribution and justify its significance. The introduction should clearly differentiate this research from prior work, highlighting its novelty to capture the reader's interest and emphasize its importance.

  "Here we applied Py-GC/MS in concert with visualization methods to assess the relative distribution of MNPs in major organ systems from human decedent livers, kidneys and brains." (Page 1)

  Implementation: Add a sentence explicitly stating the novelty of the research, such as: "This study is the first, to our knowledge, to investigate and quantify MNP presence in human brain tissue, alongside liver and kidney samples."

• Expand on Potential Health Consequences

  This medium-impact change would strengthen the introduction by providing a more complete picture of the potential health risks associated with MNPs. While the introduction mentions general health concerns, briefly elaborating on specific potential mechanisms (e.g., oxidative stress, inflammation) would provide stronger justification for the research.

  "These results highlight a critical need to better understand the routes of exposure, uptake and clearance pathways and potential health consequences of plastics in human tissues, particularly in the brain." (Page 1)

  Implementation: Add a sentence or phrase expanding on potential health consequences, such as: "...potential health consequences, including oxidative stress, inflammation, and disruption of cellular processes..."

• Clearly State Study Objective

  This low-impact improvement would provide a smoother transition between the problem statement and the study's objective. A concise sentence summarizing the study's aim would enhance the logical flow and clarity of the introduction.

  "Here we applied Py-GC/MS in concert with visualization methods to assess the relative distribution of MNPs in major organ systems from human decedent livers, kidneys and brains." (Page 1)

  Implementation: Add a sentence clearly stating the study's objective, such as: "Therefore, this study aimed to quantify MNP concentrations in human liver, kidney, and brain tissues to better understand their distribution and potential accumulation patterns."

Results

Key Aspects

• Higher MNP Concentrations in Brain Tissue: The study found significantly higher concentrations of micro- and nanoplastics (MNPs) in human brain tissue compared to liver and kidney tissues obtained from decedent samples. This finding is based on the analysis of samples from 2016 and 2024. The difference in MNP concentration between the brain and the other organs was statistically significant, as determined by a two-way ANOVA.
• Temporal Increase in MNP Concentrations: The research demonstrated a statistically significant increase in MNP concentrations in both liver and brain tissues over time, comparing samples collected in 2016 and 2024. This temporal trend suggests a potential increase in human exposure to MNPs. Statistical analysis using Mann-Whitney tests confirmed the significant difference in MNP levels between the two time points.
• Polyethylene Predominance in Brain Tissue: The study identified polyethylene (PE) as the predominant polymer type in brain tissue, with a higher proportion compared to its presence in liver and kidney samples. This finding suggests tissue-specific differences in the accumulation of different plastic polymers. The relative abundance of PE was consistent across samples and had the highest confidence spectra.
• Elevated MNP Levels in Dementia Cases: Brain samples from individuals diagnosed with dementia exhibited significantly higher MNP concentrations compared to brain tissues from individuals without dementia. This observation, while not establishing causality, suggests a potential association between MNP accumulation and neurodegenerative conditions. Additional brain samples from earlier collection years (1997-2013) showed lower MNP concentrations, further supporting the temporal trend.
• Analytical Methodology: Py-GC/MS: The study utilized pyrolysis gas chromatography–mass spectrometry (Py-GC/MS) as the primary analytical method for quantifying MNP concentrations in the tissue samples. This technique, along with visual methods, allowed for a more comprehensive and quantitative assessment of MNP presence, including smaller nanoplastics often missed by traditional microscopic methods. The consistency of Py-GC/MS data across different laboratories provides confidence in its reliability.

Strengths

• Clear Presentation of Key Findings

  The Results section clearly presents the core findings, showing significantly higher micro- and nanoplastic (MNP) concentrations in brain tissue compared to liver and kidney, and an increase in MNP concentrations in liver and brain tissues between 2016 and 2024.

  "Brain MNP concentrations were significantly higher than liver and kidney, analyzed by two-way ANOVA (P < 0.0001)...indicate significant differences in samples from the same organ between 2016 and 2024" (Page 2)
• Effective Use of Visualizations

  The section effectively uses figures and data visualizations (Fig. 1) to support the findings, making the results more accessible and understandable.

  "Fig. 1 | Overview of total MNP concentrations from all decedent samples from liver, kidney and brain." (Page 2)
• Robust Analytical Method

  The study reports the application of a robust analytical method, pyrolysis gas chromatography–mass spectrometry (Py-GC/MS), which has been validated in other studies, adding credibility to the quantification of MNPs.

  "Py-GC/MS data between labs has been comparable, providing confidence in this method for human tissue analysis" (Page 2)
• Comparison of Dementia and Non-Dementia Cases

  The study includes a comparison of MNP concentrations in brain samples from individuals with and without dementia, revealing a significant difference and suggesting a potential area for further research.

  "Brain samples from decedents with diagnosed dementia (n = 12, purple circles) from UNM exhibit far greater MNP concentrations than brain tissues from participants without dementia from New Mexico" (Page 2)
• Justification of Methodological Choice

  The section acknowledges the limitations of visual microscopic methods and justifies the use of Py-GC/MS for detecting smaller nanoplastics.

  "These methods are often limited to larger (>5 μm) particulates; thus, smaller nanoplastics are unintentionally excluded. As a new approach, pyrolysis gas chromatography–mass spectrometry (Py-GC/MS) has been applied..." (Page 2)

Suggestions for Improvement

• Report Absolute MNP Concentrations

  This high-impact improvement would significantly enhance the clarity and interpretability of the Results section. Currently, the section presents findings primarily focused on *differences* between groups (e.g., 2016 vs. 2024, brain vs. liver/kidney). However, it lacks a clear and concise statement of the *absolute* MNP concentrations found in each tissue type. This omission makes it difficult for readers to grasp the overall magnitude of MNP presence and compare the findings to other studies or exposure scenarios. The Results section is the appropriate place for this information, as it's where the core quantitative data are presented.

  "Fig. 1 | Overview of total MNP concentrations from all decedent samples from liver, kidney and brain. a, Microplastic concentrations in liver, kidney and brain decedent human samples (n = 20–28 separate participants for each timepoint; Supplementary Table 1) from the UNM OMI." (Page 2)

  Implementation: Add a sentence or short paragraph at the beginning of the Results section summarizing the overall range and central tendency (e.g., median or mean) of MNP concentrations found in each tissue type (brain, liver, kidney). For example: "Across all samples, median MNP concentrations were X µg/g in brain tissue, Y µg/g in liver tissue, and Z µg/g in kidney tissue. The range of concentrations observed was A-B µg/g in brain, C-D µg/g in liver, and E-F µg/g in kidney."

• Explicitly State Sample Sizes in Text

  This medium-impact change would improve the completeness and clarity of the Results section. While the section mentions the analysis of samples from different years (2016 and 2024), it doesn't explicitly state the *number* of samples analyzed in each group for each tissue type. This information is crucial for assessing the statistical power and robustness of the comparisons. This belongs in the results section, as it provides context for the quantitative data.

  "Microplastic concentrations in liver, kidney and brain decedent human samples (n = 20–28 separate participants for each timepoint; Supplementary Table 1)" (Page 2)

  Implementation: Include the sample sizes (n values) for each group (2016 and 2024) and each tissue type (brain, liver, kidney) directly in the text, not just in the figure legend. For example: "Liver samples from 2016 (n=X) and 2024 (n=Y) were analyzed..."

• Improve Organizational Structure

  This medium-impact improvement would enhance the clarity and flow of the Results section. The section jumps between different comparisons (tissue types, time points, dementia status) without clear transitions or a logical organizational structure. This makes it difficult for the reader to follow the progression of findings. Improving the organization would make the results easier to understand and interpret. The results section should have a logical flow.

  "Fig. 1 | Overview of total MNP concentrations from all decedent samples from liver, kidney and brain." (Page 2)

  Implementation: Organize the Results section into subsections with clear headings that reflect the different comparisons being made. For example: * "MNP Concentrations in Different Tissue Types" * "Temporal Trends in MNP Concentrations" * "MNP Concentrations in Dementia vs. Non-Dementia Brain Samples"

• List All Analyzed Polymers

  This low-impact improvement would enhance the clarity of the Results section. While the section mentions "12 different polymers," it doesn't list them all in the main text. Listing these polymers would provide a more complete picture of the MNP composition and allow for easier comparison with other studies. This is relevant to the results as it provides context for the data.

  "b, Overall distribution of 12 different polymers suggests a greater accumulation of PE in the brain relative to liver or kidney" (Page 2)

  Implementation: List the 12 different polymers analyzed in the main text of the Results section, not just in the figure legend or methods. For example: "The analysis included 12 different polymers: polyethylene (PE), polypropylene (PP), ..."

• Mention Orthogonal Methods

  This low-impact improvement would add clarity. The section mentions "orthogonal methods" but doesn't explicitly name them in the Results. While they are likely detailed in the Methods, briefly mentioning them here would provide context for the Py-GC/MS results. This is relevant to the results as it reinforces the robustness of the findings.

  "when coupled with orthogonal methods. Py-GC/MS data between labs has been comparable, providing confidence in this method for human tissue analysis" (Page 2)

  Implementation: Briefly mention the orthogonal methods used alongside Py-GC/MS in the Results section. For example: "Consistent with the Py-GC/MS findings, analysis using [orthogonal method 1] and [orthogonal method 2] also showed..."

Non-Text Elements

Fig. 1 | Overview of total MNP concentrations from all decedent samples from...

Full Caption

Fig. 1 | Overview of total MNP concentrations from all decedent samples from liver, kidney and brain. a, Microplastic concentrations in liver, kidney and brain decedent human samples (n = 20–28 separate participants for each timepoint; Supplementary Table 1) from the UNM OMI. Data are shown on a log10 scale, with the bar representing the group median value and 95% confidence interval. Orange-colored symbols in the 2016 brain samples were analyzed independently at Oklahoma State University. P values from Mann-Whitney tests (two-sided) indicate significant differences in samples from the same organ between 2016 and 2024 (with more comprehensive statistical treatments in Supplementary Methods-Statistical analysis). Brain MNP concentrations were significantly higher than liver and kidney, analyzed by two-way ANOVA (P < 0.0001). b, Overall distribution of 12 different polymers suggests a greater accumulation of PE in the brain relative to liver or kidney (average shown per group; see Extended Data Fig. 1 for individual data). c, PE (which was in the highest abundance and consistently had the highest confidence spectra) concentrations in all organs followed similar trends compared to total plastics (also represented as group median value and

Figure/Table Image (Page 2)

First Reference in Text

Py-GC/MS measurements of MNP concentrations in decedent liver and kidney specimens were similar, with the median value of total plastics at 433 and 404 µg g⁻¹, respectively, from 2024 samples (Fig. 1a and Supplementary Table 1).

Description

• Microplastic concentrations and statistical analysis: Figure 1 presents an overview of microplastic and nanoplastic (MNP) concentrations in human tissues. Panel a shows the concentrations of microplastics in liver, kidney, and brain samples obtained from deceased individuals. These samples were collected at two different time points: 2016 and 2024. The number of participants for each time point ranges from 20 to 28. These samples were part of a study conducted at the University of New Mexico's Office of the Medical Investigator (UNM OMI). The data is displayed on a log10 scale, which means that each increment on the y-axis represents a tenfold increase in concentration. The bar in each group represents the median value, which is the midpoint of the data when arranged in order, and the error bars indicate a 95% confidence interval, providing a range within which the true population median is likely to fall. The data for the 2016 brain samples, shown in orange, were analyzed separately at Oklahoma State University. The statistical significance of differences between samples from the same organ collected in 2016 versus 2024 was assessed using a Mann-Whitney test, a statistical test used to compare two independent groups when the data is not normally distributed. The p-value indicates the probability of observing the results if there is no actual difference between the groups; a smaller p-value suggests a stronger evidence against the null hypothesis (no difference). A two-way ANOVA (analysis of variance) was used to analyze whether brain MNP concentrations were significantly different from liver and kidney concentrations. ANOVA is a statistical test that can detect differences between the means of two or more groups. The caption reports that the p-value for this comparison was less than 0.0001, indicating a very statistically significant difference.
• Polymer distribution: Panel b shows the overall distribution of 12 different polymers (types of plastics) found in the samples. This distribution is presented as the average percentage of each polymer's mass within each organ (liver, kidney, and brain). The figure suggests that polyethylene (PE) accumulates more in the brain compared to the liver or kidney. The individual data for this panel can be found in Extended Data Figure 1.
• Polyethylene concentrations: Panel c displays the concentrations of polyethylene (PE) in all organs, following similar trends to the total plastic concentrations. Polyethylene (PE) had the highest abundance and the most reliable spectral data. The concentrations are shown as group median values, similar to panel a. The p-values are calculated using the Mann-Whitney test.

Scientific Validity

• Justification of statistical tests: The use of Mann-Whitney tests for comparing MNP concentrations between 2016 and 2024 is appropriate given that MNP concentration data may not be normally distributed. However, the caption should explicitly state whether data were tested for normality and justify the choice of non-parametric tests if normality assumptions were violated.
• Consideration of confounding factors: The ANOVA results indicating significantly higher MNP concentrations in the brain compared to the liver and kidney are compelling. However, it's important to consider potential confounding factors, such as differences in tissue processing or analytical sensitivity across different organs. These potential confounders should be addressed in the discussion.
• Definition of 'confidence spectra': The statement that PE had the highest abundance and consistently had the highest confidence spectra is important for justifying the focus on PE in panel c. However, the criteria for determining 'confidence spectra' should be clearly defined in the methods or supplementary information.

Communication

• Caption length and density: The caption is comprehensive, providing a good overview of what each panel of Figure 1 represents. However, it's quite dense, and breaking it into shorter sentences might improve readability.
• Justification of statistical tests: While the caption mentions the use of statistical tests, it might be beneficial to briefly state the rationale for choosing those specific tests (Mann-Whitney and two-way ANOVA) in the context of the data's characteristics.
• Clarity of references to other figures: Referencing Extended Data Fig. 1 in panel (b) is appropriate, but consider whether a very brief description of what this figure shows could be incorporated into the caption for added clarity.

Fig. 2 | Visualization of putative plastics in the brain. a, b, Polarization...

Full Caption

Fig. 2 | Visualization of putative plastics in the brain. a, b, Polarization wave microscopy (a, black arrows indicate refractory inclusions; inset is a digital magnification for clarity) and SEM (b, visual fields are 15.4 and 20.1 µm wide) were used to scan sections of brain from decedent human samples. c, Large (>1 µm) inclusions were not observed; additional polarization wave examples are highlighted (white arrows highlight submicron refractory inclusions). Resolution limitations of these technologies drove the use of TEM to examine the extracts from the pellets used for Py-GC/MS. d, Example TEM images resolved

Figure/Table Image (Page 4)

First Reference in Text

Using scanning electron microscopy (SEM) and polarization wave microscopy, refractory inclusions were identified in all organs histo-logically (Fig. 2, Extended Data Fig. 3 and Supplementary Figs. 7-16).

Description

• Polarization wave microscopy and SEM: Figure 2 presents visual evidence of potential plastic particles within brain tissues. Panels a and b show images obtained using two different types of microscopy: polarization wave microscopy and scanning electron microscopy (SEM). Polarization wave microscopy uses polarized light to identify materials that are birefringent, meaning they split light into two rays with different refractive indices, often used to visualize crystalline materials. In the image (panel a), black arrows point to 'refractory inclusions,' which are materials that resist changes, indicating they might be foreign particles. The inset in panel a provides a magnified view for better clarity. Panel b shows a scanning electron microscopy (SEM) image, where electrons scan the surface of the sample to create a detailed image. The visual fields for the SEM images are 15.4 and 20.1 micrometers wide, which gives a sense of the scale being observed.
• Resolution limitations and submicron inclusions: Panel c notes that large inclusions, specifically those larger than 1 micrometer (a unit of length equal to one-millionth of a meter), were not observed in the samples. The panel highlights additional examples of polarization wave microscopy images, with white arrows indicating submicron refractory inclusions, which are inclusions smaller than 1 micrometer. The text mentions that the resolution limitations of polarization wave microscopy and SEM led the researchers to use transmission electron microscopy (TEM).
• Transmission electron microscopy (TEM): Panel d shows example images obtained using transmission electron microscopy (TEM). TEM involves passing a beam of electrons through a very thin sample to create an image, allowing for much higher resolution than light microscopy or SEM. This technique was used to examine extracts from the pellets that were also used for pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS), a method used to identify the chemical composition of the sample. The TEM images are meant to resolve the nature of the particles identified by the other methods.

Scientific Validity

• Need for spectroscopic confirmation: The use of multiple microscopy techniques (polarization wave microscopy, SEM, and TEM) provides complementary evidence for the presence of putative plastics. However, it's crucial to acknowledge that morphological identification alone is insufficient for definitive identification of plastic particles. Spectroscopic confirmation (e.g., Raman spectroscopy, FTIR) is needed to confirm the chemical composition of the observed inclusions.
• Quantification and detection limits: The caption mentions that 'large (>1 μm) inclusions were not observed,' which is an important negative finding. However, it would be helpful to quantify the number of samples examined by each technique and to provide an estimate of the detection limits for each method.
• Representativeness of TEM results: The statement that 'resolution limitations of these technologies drove the use of TEM' is valid. However, it's important to acknowledge that TEM analysis is typically performed on a small subset of samples due to its time-consuming nature. Therefore, the TEM results may not be fully representative of the entire sample set.

Communication

• Rationale for imaging techniques: The caption effectively describes the progression of imaging techniques used, from polarization wave microscopy and SEM to TEM. However, it would be helpful to explicitly state the rationale for using each technique and how they complement each other in addressing the research question.
• Explanation of TEM's advantages: While the caption mentions the limitations of polarization wave microscopy and SEM in resolving submicron inclusions, it could be strengthened by briefly explaining why TEM is better suited for this purpose (e.g., higher resolution).
• Description of refractory inclusions: The use of arrows to indicate refractory inclusions in the polarization wave microscopy images is clear. However, it might be helpful to provide a brief description of what these inclusions are and why they are considered 'putative plastics.'

Discussion

Key Aspects

• Increasing Environmental Concentrations and Internal Accumulation: The study hypothesizes that the increasing environmental concentrations of micro- and nanoplastics (MNPs) may lead to a corresponding increase in internal MNP concentrations within the human body. This is based on the observation of a roughly 50% increase in plastic concentrations in the brains analyzed over the past 8 years. This hypothesis connects the study's findings to the broader issue of environmental pollution and its potential impact on human health.
• Potential Mechanisms of MNP Uptake and Distribution: The Discussion explores potential mechanisms of MNP uptake and distribution within the body, particularly focusing on how nanoplastics might reach the brain. It draws parallels with studies in *Daphnia magna*, suggesting clathrin-dependent endocytosis and macropinocytosis as potential mechanisms. It also proposes that ingestion of lipids might facilitate the transfer of MNPs into the brain, although the exact pathways remain unknown.
• Consideration of Decedent Tissue Limitations: The Discussion acknowledges the limitations of using decedent tissues, noting that while blood was not cleared from the organs during autopsy, it's unlikely that the nanoplastics in the brain are solely from the vascular compartment. This is supported by the observation of comparable blood volumes in the kidneys and livers. This consideration of potential confounding factors adds to the study's scientific rigor.
• Non-linear Relationship with Age and Potential Equilibrium: The section discusses the lack of correlation between total plastic concentrations and decedent age, suggesting that MNP accumulation may not be a simple linear function of lifespan. Instead, it proposes that internal MNP concentrations might reach an equilibrium, influenced by exposure, uptake, and clearance rates. This highlights the complexity of MNP bioaccumulation and the need for further research on clearance mechanisms.
• Methodological Limitations and Contamination Concerns: The Discussion addresses the limitations of the study, including the use of new analytical methods (Py-GC/MS) that are not yet widely standardized. It also acknowledges the potential for external contamination, although numerous quality control steps were taken to minimize this. The section also notes the within-sample coefficient of variation and the lack of focus on minimizing external plastic contamination during specimen collection in the past.
• Potential for Overestimation or Underestimation: The Discussion highlights the potential for overestimation or underestimation of polymer mass concentrations due to factors such as residual biomatrix interference in Py-GC/MS analysis and incomplete collection of nanoplastics during ultracentrifugation. It also mentions the possibility of advanced oxidative degradation of MNPs, leading to an underestimation of concentrations. These considerations add nuance to the interpretation of the quantitative data.

Strengths

• Connection to Broader Context

  The Discussion effectively connects the study's findings back to the broader context of increasing environmental MNP concentrations and the potential implications for human health.

  "we postulate that the exponentially increasing environmental concentrations of MNPs2,14 may analogously increase internal maximal concentrations." (Page 3)
• Acknowledgement of Limitations

  The section acknowledges the limitations of the study, such as the lack of standardized analytical methods and the potential for contamination, which adds to the scientific rigor and transparency.

  "Although the current data derive from multiple tissue banks and two analytic sites replicating key results, the new analytical Py-GC/ MS methods applied here are yet to be widely adopted and refined into standardized tests for clinical specimens." (Page 3)
• Highlighting of Novelty

  The Discussion highlights the novelty of the findings, particularly the detection of MNPs in human brain tissue and the higher concentrations observed in dementia cases.

  "Py-GC/MS analysis revealed total plastics concentrations in dementia samples...that were higher than in any normal frontal cortex cohort" (Page 3)
• Discussion of Potential Mechanisms

  The section discusses potential mechanisms of MNP uptake and distribution, drawing on insights from other organisms and suggesting possible pathways in humans.

  "Insights from Daphnia magna suggest clathrin-dependent endocytosis and macropinocytosis may underlie nanoplastic translocation within the intestine13; we posit a similar uptake may occur in human ingestion of lipids that would also facilitate selective transfer into the brain." (Page 3)
• Caution Regarding Causality

  The section appropriately emphasizes that the findings regarding dementia are associative and do not establish causality, maintaining scientific accuracy and avoiding overinterpretation.

  "MNP uptake and distribution pathways are poorly understood...thus, no causality is assumed from these findings." (Page 3)

Suggestions for Improvement

• Expand Literature Comparison

  This high-impact improvement would significantly enhance the Discussion's depth and impact by placing the current findings within the context of existing literature on MNP exposure and potential health effects. The Discussion section's purpose is to interpret the results in a broader scientific context. Currently, the Discussion makes limited references to other studies, primarily focusing on methodological comparisons. A more thorough comparison with existing research would strengthen the study's conclusions and highlight its contribution to the field.

  "Although there are few studies to draw on yet performed in mammals, in zebrafish exposed to constant concentrations, nanoplastic uptake increased to a stable plateau and cleared after exposure15" (Page 3)

  Implementation: Incorporate a more comprehensive discussion of relevant literature on MNP exposure, tissue distribution, and potential health effects (especially neurological effects). Compare and contrast the current findings with those of other studies, highlighting areas of agreement, disagreement, and novelty. For example: "Our findings of PE predominance in the brain are consistent with [citation] who reported... However, our observation of higher MNP concentrations in dementia cases contrasts with [citation] who found... This discrepancy may be due to..."

• Elaborate on Potential Biases

  This medium-impact improvement would strengthen the Discussion by providing a more balanced perspective on the study's limitations. The Discussion section should critically evaluate the study's strengths and weaknesses. While the Discussion acknowledges some limitations (e.g., lack of standardized methods), it could be more explicit about potential biases or confounding factors that might influence the results. This is crucial for a balanced interpretation of the findings.

  "Although the current data derive from multiple tissue banks and two analytic sites replicating key results, the new analytical Py-GC/ MS methods applied here are yet to be widely adopted and refined into standardized tests for clinical specimens." (Page 3)

  Implementation: Expand the discussion of limitations to include potential biases or confounding factors, such as: * The use of decedent tissues, which may not fully reflect MNP distribution in living individuals. * The potential for postmortem changes in MNP distribution or degradation. * The limited demographic information available for the samples, which restricts the ability to control for potential confounders. For example: "It is important to acknowledge that the use of decedent tissues may introduce certain biases. Postmortem changes in tissue integrity or MNP distribution cannot be entirely ruled out..."

• Provide Specific Future Research Directions

  This medium-impact change would enhance the Discussion by providing a more concrete and actionable roadmap for future research. The Discussion section should outline future research directions. While the Discussion mentions the need for further research, it could be more specific about the types of studies needed to address the remaining knowledge gaps. This would provide a clearer direction for the field.

  "For this, refinements to the analytical techniques, more complex study designs and much larger cohorts are needed." (Page 3)

  Implementation: Provide more specific recommendations for future research, such as: * Studies investigating the mechanisms of MNP uptake and transport across the blood-brain barrier. * Longitudinal studies examining the relationship between MNP exposure and neurological outcomes in living individuals. * Research to develop and validate standardized methods for MNP detection and quantification in human tissues. * Studies exploring the potential role of MNP exposure in the pathogenesis of specific neurodegenerative diseases. For example: "Future research should focus on elucidating the mechanisms by which MNPs cross the blood-brain barrier. Longitudinal studies with well-defined cohorts are needed to investigate the potential link between chronic MNP exposure and the development of neurodegenerative diseases..."

• Improve Organizational Structure

  This low-impact improvement would improve the clarity and flow of the Discussion. The section jumps between different topics (limitations, mechanisms, future research) without clear transitions or a strong unifying narrative. A more organized structure would enhance readability and strengthen the overall argument.

  "While we suspected that MNPs might accumulate in the body over a lifespan, the lack of correlation between total plastics and decedent age (P = 0.87 for brain data) does not support this (Supplementary Fig. 1)." (Page 3)

  Implementation: Organize the Discussion into subsections with clear headings that reflect the different themes being addressed. For example: * "Comparison with Existing Literature" * "Potential Mechanisms of MNP Uptake and Distribution" * "Limitations of the Current Study" * "Future Research Directions" * "Conclusions"

• Discuss Alternative Uptake Pathways

  This low-impact improvement would add nuance to the discussion of potential MNP accumulation mechanisms. The Discussion mentions lipid ingestion as a potential pathway, but it could briefly discuss other possibilities, such as inhalation or uptake through the olfactory system, to provide a more complete picture.

  "we posit a similar uptake may occur in human ingestion of lipids that would also facilitate selective transfer into the brain." (Page 3)

  Implementation: Briefly mention other potential routes of MNP exposure and uptake into the brain, such as inhalation and subsequent translocation via the olfactory nerve. For example: "In addition to ingestion, inhalation of airborne MNPs may represent another route of exposure. Studies have suggested that inhaled nanoparticles can reach the brain via the olfactory nerve [citation]..."

Methods

Key Aspects

• Human Tissue Sample Collection: Human tissue samples (brain, liver, and kidney) were obtained from autopsies conducted at the UNM OMI, following a consistent tissue collection protocol. Samples from 2016 and 2024 were collected and stored in 10% formalin. Additionally, brain samples from individuals with confirmed dementia were included, and further brain samples were obtained from repositories on the East Coast to extend the range of death years back to 1997.
• Py-GC/MS Detection of Polymer Solids: Pyrolysis gas chromatography-mass spectrometry (Py-GC/MS) was employed to detect and quantify polymer solids in the tissue samples. This method involves isolating solid particulates from chemically digested tissue, followed by combustion to reveal signature mass spectra for 12 specific polymers: PE, PVC, nylon 66, SBR, acrylonitrile butadiene styrene, polyethylene terephthalate, nylon-6, poly(methyl methacrylate), polyurethane, polycarbonate, PP and polystyrene. This technique allows for the identification and quantification of micro- and nanoplastics in complex biological matrices.
• Sample Preparation: Digestion and Ultracentrifugation: Tissue samples (approximately 500 mg) were digested with 10% potassium hydroxide to remove biological material. The digested samples were then ultracentrifuged to generate a pellet enriched in solid materials, including polymer-based solids. A small portion of this pellet (1-2 mg) was analyzed by single-shot Py-GC/MS.
• Data Analysis and Normalization: The Py-GC/MS data were analyzed by comparing the obtained spectra to a microplastics-CaCO3 standard containing the 12 specified polymers. Polymer spectra were identified using F-Search MPs v2.1 software. The resulting data were normalized to the original sample weight to obtain a mass concentration of microplastics in micrograms per gram of tissue (µg g-1).
• Reference to Supplementary Methods: The Methods section references supplementary methods for detailed information on the Py-GC/MS procedures and statistical analyses. This includes details on Py-GCMS operating settings, polymer pyrolyzate targets, and examples of spectra. This approach allows for a concise presentation of the core methods in the main text while providing comprehensive details in the supplementary materials.

Strengths

• Clear Description of Tissue Sources

  The Methods section clearly describes the source of human tissue samples, including the use of a consistent collection protocol and the inclusion of samples from multiple repositories.

  "The same tissue collection protocol at the UNM OMI was used for 2016 and 2024. Small pieces of representative organs (3 5 cm3) were routinely collected at autopsy and stored in 10% formalin." (Page 7)
• Concise Overview of Py-GC/MS Method

  The section provides a concise overview of the Py-GC/MS method used for detecting polymer solids, referencing detailed supplementary methods for further information.

  "Py-GC/MS is an informative and reliable method to determine plastic concentrations in liquid and solid tissue samples, with ample assurance of accuracy, quality and rigor3,4,9,10." (Page 7)
• Specification of Analyzed Polymers

  The section clearly states the specific polymers included in the microplastics-CaCO3 standard used for comparison in the Py-GC/MS analysis.

  "A 12 mg portion of the resulting pellet was then analyzed by single-shot Py-GC/MS and compared to a microplastics-CaCO3 standard containing the following 12 specific polymers: PE, PVC, nylon 66, SBR, acrylonitrile butadiene styrene, polyethylene terephthalate, nylon-6, poly(methyl methacrylate), polyurethane, polycarbonate, PP and polystyrene." (Page 7)
• Identification of Analysis Software

  The section mentions the software used for identifying polymer spectra, adding to the transparency and reproducibility of the analysis.

  "Polymer spectra were identified via F-Search MPs v2.1 software (Frontier Labs)." (Page 7)
• Mention of Data Normalization

  The section briefly mentions the data normalization step, which is crucial for accurate quantification of MNP concentrations.

  "The resulting data were normalized to the original sample weight to render a mass concentration (g g1)." (Page 7)

Suggestions for Improvement

• Provide Detailed Tissue Collection Protocol

  This high-impact improvement would greatly enhance the study's methodological rigor and transparency. The Methods section is the appropriate location for this level of detail. Currently, the Methods section provides a general overview of the tissue collection protocol but lacks crucial details about the *specific* procedures used to ensure consistency and minimize contamination. This omission undermines the reliability of the findings, as variations in tissue handling could significantly impact MNP measurements. Providing a detailed, step-by-step protocol is essential for reproducibility and allows other researchers to critically evaluate the methods.

  "The same tissue collection protocol at the UNM OMI was used for 2016 and 2024. Small pieces of representative organs (35 cm3) were routinely collected at autopsy and stored in 10% formalin." (Page 7)

  Implementation: Provide a detailed, step-by-step protocol for tissue collection, including: * Specific anatomical locations from which tissues were sampled (beyond just "frontal cortex"). * Instruments used for tissue dissection and handling (e.g., type of scalpel, forceps). * Detailed description of the containers used for tissue storage (material, size, cleaning procedures). * Specific steps taken to minimize contamination during collection and handling (e.g., use of gloves, sterile instruments, clean room environment). * Duration and conditions of tissue storage before processing. Example: "Brain tissue samples (approximately 1 cm³) were obtained from the left superior frontal gyrus using a sterile, disposable stainless steel scalpel and PTFE-coated forceps. Samples were immediately placed in pre-cleaned, 50 mL polypropylene containers filled with 10% neutral buffered formalin..."

• Detail Contamination Control Procedures

  This high-impact improvement is crucial for ensuring the reproducibility and validity of the study's findings. The Methods section is the appropriate place to detail these critical steps. The current Methods section describes the chemical digestion and ultracentrifugation steps but lacks crucial details about the *specific* procedures and quality control measures used to prevent and monitor contamination. This omission raises concerns about the potential for external MNP contamination to influence the results. A detailed description of contamination control measures is essential for demonstrating the rigor of the study.

  "Samples (~500 mg) were digested with 10% potassium hydroxide for at least 3 days at 40 C. Samples were then ultracentrifuged at 100,000g for 4 h to generate a pellet enriched in solid materials resistant to such digestion, which included polymer-based solids10." (Page 7)

  Implementation: Provide a detailed description of the contamination control procedures, including: * Specific measures taken to prevent contamination during sample processing (e.g., use of laminar flow hood, filtered solutions, pre-cleaned glassware). * Detailed description of blank samples used (e.g., reagent blanks, procedural blanks) and their processing. * Frequency of blank sample analysis and acceptance criteria for blank contamination levels. * Specific steps taken to clean and prepare glassware and equipment. * Details of any air filtration or environmental monitoring used in the laboratory. Example: "All sample preparation steps were performed in a Class 100 laminar flow hood. Reagents were filtered through 0.2 µm PTFE filters before use. Glassware was thoroughly cleaned with [detergent], rinsed with ultrapure water, and baked at [temperature] for [duration]. Procedural blanks (containing all reagents but no tissue) were processed alongside each batch of samples..."

• Specify Py-GC/MS Instrument Parameters

  This medium-impact improvement would enhance the clarity and reproducibility of the Py-GC/MS analysis. The Methods section should provide all necessary details. While the Methods section mentions Py-GC/MS, it lacks specific details about the *instrument parameters* used. This omission makes it difficult for other researchers to replicate the analysis and compare their findings. Providing these details is standard practice in scientific publications and enhances the study's transparency.

  "A 12 mg portion of the resulting pellet was then analyzed by single-shot Py-GC/MS and compared to a microplastics-CaCO3 standard..." (Page 7)

  Implementation: Provide specific details about the Py-GC/MS instrument parameters, including: * Make and model of the Py-GC/MS instrument. * Pyrolysis temperature and duration. * GC column type and dimensions. * GC oven temperature program. * Carrier gas and flow rate. * MS detector type and settings (e.g., scan range, ionization mode). Example: "Py-GC/MS analysis was performed using a [Make and Model] instrument equipped with a [Pyrolyzer Model] pyrolyzer. Pyrolysis was conducted at [temperature] for [duration]. The GC separation was performed using a [Column Type] column ([dimensions]) with a [Carrier Gas] carrier gas at a flow rate of [flow rate]..."

• Specify Units for Sample Weight

  This medium-impact improvement would improve the clarity and completeness of the Methods section. It is important to specify how data were handled. The Methods section mentions that the resulting data were normalized to the original sample weight, but it doesn't specify the *units* used for the sample weight. This ambiguity could lead to confusion and inconsistencies in data interpretation. Specifying the units is a simple but important detail that enhances the clarity and reproducibility of the study.

  "The resulting data were normalized to the original sample weight to render a mass concentration (g g1)." (Page 7)

  Implementation: Specify the units used for the original sample weight in the data normalization step. Example: "The resulting data were normalized to the original sample weight (in grams) to render a mass concentration (µg g-1)."

• Clarify Recording of Cause of Death

  This low-impact improvement would enhance the completeness of the Methods section. It is important to fully describe the methods used. While the section mentions that limited demographic data were available, it doesn't explicitly state whether *cause of death* was recorded for all participants or only a subset. Clarifying this point would provide a more complete picture of the available data and potential limitations.

  "Limited demographic data (age, sex, race/ethnicity, cause of death and date of death) were available due to the conditions of specimen approval..." (Page 7)

  Implementation: Clarify whether cause of death was recorded for all participants or only a subset. Example: "Limited demographic data (age, sex, race/ethnicity, cause of death [recorded for all participants], and date of death) were available..."