Parkinson’s disease (PD) is an incurable, devastating disorder that is characterized by pathological protein aggregation and neurodegeneration in the substantia nigra. In recent years, growing evidence has implicated the gut environment and the gut-brain axis in the pathogenesis and progression of PD, especially in a subset of people who exhibit prodromal gastrointestinal dysfunction. Specifically, perturbations of gut homeostasis are hypothesized to contribute to α-synuclein aggregation in enteric neurons, which may spread to the brain over decades and eventually result in the characteristic central nervous system manifestations of PD, including neurodegeneration and motor impairments. However, the mechanisms linking gut disturbances and α-synuclein aggregation are still unclear. A plethora of research indicates that toll-like receptors (TLRs), especially TLR2 and TLR4, are critical mediators of gut homeostasis. Alongside their established role in innate immunity throughout the body, studies are increasingly demonstrating that TLR2 and TLR4 signalling shapes the development and function of the gut and the enteric nervous system. Notably, TLR2 and TLR4 are dysregulated in patients with PD, and may thus be central to early gut dysfunction in PD. To better understand the putative contribution of intestinal TLR2 and TLR4 dysfunction to early α-synuclein aggregation and PD, we critically discuss the role of TLR2 and TLR4 in normal gut function as well as evidence for altered TLR2 and TLR4 signalling in PD, by reviewing clinical, animal model and in vitro research. Growing evidence on the immunological aetiology of α-synuclein aggregation is also discussed, with a focus on the interactions of α-synuclein with TLR2 and TLR4. We propose a conceptual model of PD pathogenesis in which microbial dysbiosis alters the permeability of the intestinal barrier as well as TLR2 and TLR4 signalling, ultimately leading to a positive feedback loop of chronic gut dysfunction promoting α-synuclein aggregation in enteric and vagal neurons. In turn, α-synuclein aggregates may then migrate to the brain via peripheral nerves, such as the vagal nerve, to contribute to neuroinflammation and neurodegeneration typically associated with PD.

The health and well-being of retired rugby union and league players, particularly regarding the long-term effects of concussions, are of major concern. Concussion has been identified as a major risk factor for neurodegenerative diseases, such as Alzheimer’s and Amyotrophic Lateral Sclerosis (ALS), in athletes engaged in contact sports. This study aimed to assess differences in specific biomarkers between UK-based retired rugby players with a history of concussion and a non-contact sports group, focusing on biomarkers associated with Alzheimer’s, ALS, and CTE. We randomly selected a sample of male retired rugby or non-contact sport athletes (n = 56). The mean age was 41.84 ± 6.44, and the mean years since retirement from the sport was 7.76 ± 6.69 for participants with a history of substantial concussions (>5 concussions in their career) (n = 30). The mean age was 45.75 ± 11.52, and the mean years since retirement was 6.75 ± 4.64 for the healthy controls (n = 26). Serum biomarkers (t-tau, RBP-4, SAA, Nf-L, and retinol), plasma cytokines, and biomarkers associated with serum-derived exosomes (Aβ42, p-tau181, p-tau217, and p-tau231) were analyzed using validated commercial ELISA assays. The results of the selected biomarkers were compared between the two groups. Biomarkers including t-tau and p-tau181 were significantly elevated in the history of the substantial concussion group compared to the non-contact sports group (t-tau: p < 0.01; p-tau181: p < 0.05). Although between-group differences in p-tau217, p-tau231, SAA, Nf-L, retinol, and Aβ42 were not significantly different, there was a trend for higher levels of Aβ42, p-tau217, and p-tau231 in the concussed group. Interestingly, the serum-derived exosome sizes were significantly larger (p < 0.01), and serum RBP-4 levels were significantly reduced (p < 0.05) in the highly concussed group. These findings indicate that retired athletes with a history of multiple concussions during their careers have altered serum measurements of exosome size, t-tau, p-tau181, and RBP-4. These biomarkers should be explored further for the prediction of future neurodegenerative outcomes, including ALS, in those with a history of concussion.

Mild traumatic brain injury (mTBI) causes structural, cellular and biochemical alterations which are difficult to detect in the brain and may persist chronically following single or repeated injury. Lipids are abundant in the brain and readily cross the blood-brain barrier, suggesting that lipidomic analysis of blood samples may provide valuable insight into the neuropathological state. This study used liquid chromatography-mass spectrometry (LC-MS) to examine plasma lipid concentrations at 11 days following sham (no injury), one (1×) or two (2×) mTBI in rats. Eighteen lipid species were identified that distinguished between sham, 1× and 2× mTBI. Three distinct patterns were found: (1) lipids that were altered significantly in concentration after either 1× or 2× F mTBI: cholesterol ester CE (14:0) (increased), phosphoserine PS (14:0/18:2) and hexosylceramide HCER (d18:0/26:0) (decreased), phosphoinositol PI(16:0/18:2) (increased with 1×, decreased with 2× mTBI); (2) lipids that were altered in response to 1× mTBI only: free fatty acid FFA (18:3 and 20:3) (increased); (3) lipids that were altered in response to 2× mTBI only: HCER (22:0), phosphoethanolamine PE (P-18:1/20:4 and P-18:0/20:1) (increased), lysophosphatidylethanolamine LPE (20:1), phosphocholine PC (20:0/22:4), PI (18:1/18:2 and 20:0/18:2) (decreased). These findings suggest that increasing numbers of mTBI induce a range of changes dependent upon the lipid species, which likely reflect a balance of damage and reparative responses.

Cellular responses at the sub-acute phase of mild traumatic brain injury (mTBI), and their contribution to ongoing damage, are unclear, complex and require simultaneous assessment of multiple cells to elucidate. An 11-colour flow cytometry method for analysing brain cells was evaluated in a weight-drop rat model of repeated mTBI. Animals received sham, one, two or three mTBI delivered at 24 h intervals (n = 6/group). Cerebrum homogenates were prepared 11 days after first mTBI, in two cohorts of n = 3/group to enable same-day staining of fresh tissue. Percentages of neurons, astrocytes, microglia, mature oligodendrocytes and NeuN + CC1+ cells, neutrophils, macrophages and non-myeloid leukocytes, and their immunoreactivity for cell damage indicators (inducible nitric oxide synthase; iNOS, proliferating cell nuclear antigen; PCNA, 8-Oxo-2'-deoxyguanosine; 8OHDG and 4-hydroxynonenal; HNE), were assessed. Median fluorescence intensity (MFI) of iNOS in activated microglia increased following two, but not one or three, mTBI (p = 0.04). However, there were differences between processing cohorts in terms of percentages and MFI of some PCNA+, iNOS+, 8OHDG + and HNE + cell populations. Previous applications of flow cytometry for rat brain analysis were typically limited to three or four markers. This method uses 11 markers to identify nine cell populations and evaluate their immunoreactivity to four metabolic indicators of cell damage. Flow cytometry can be useful for discerning injury-related changes in multiple rat brain cells. However, markers sensitive to subtle changes in experimental conditions must be identified in pilot experiments and subsequently analysed in the same tissue-processing cohort.

Humans are chronically exposed to microplastics (plastic particles < 1 mm) via oral ingestion. Yet, intestinal absorption of microplastics requires robust quantification and in vitro studies have largely lacked digestive conditions in experimental design. This study aimed to quantify how simulated digestion of microplastics affected the rate of internalisation by an intestinal epithelial cell line (IEC-6). Spherical 0.56 µm polystyrene microplastics were simulatively digested using a static digestion protocol. IEC-6 cells were exposed to 1 µg/mL of pristine or digested microplastics for 24 h. Microplastic internalisation was visualised with confocal microscopy and novelly quantified by imaging flow cytometry. Confocal microscopy confirmed internalisation of pristine and digested microplastics by IEC-6 cells, where pristine microplastics were internalised by 62.2 ± 11.8 % of cells which was lessened to 24.6 ± 5.3 % for digested microplastics. Most IEC-6 cells internalised 1-2 microplastics, and internalisation of > 2 microplastics per cell was increased (p < 0.01) after exposure to pristine microplastics (34.3 ± 10.3 %) compared to digested microplastics (8.9 ± 0.74 %). This study demonstrated that simulated digestion of polystyrene microplastics reduced internalisation by IEC-6 cells, providing unprecedented insights into the gastrointestinal effect of microplastic exposure and raising important considerations for future research.

Microplastics are an environmental and dietary contaminant of significant concern for human health due to chronic exposure and emerging evidence of cellular impact. However, understanding of this impact is limited by challenges in their detection and identification in biological samples. This study aimed to use Optical Photothermal Infrared Spectroscopy (O-PTIR) to detect and characterize real-world microplastics internalized by intestinal cells, and to investigate the cellular impact of such exposure. An intestinal epithelial cell line (IEC-6) was exposed to physically degraded fluorescent microplastics (25-100 μg/mL) made of four polymers for 24 h. O-PTIR reference spectra were acquired on single dyed primary plastics before fluorescent guided O-PTIR was used to identify and characterize microplastics in IEC-6 cells. We determined that the limit of resolution under these conditions was particles of ∼ 1 μm. Microplastics from all polymer types were successfully detected and discriminated using O-PTIR data alone. Importantly, principal component analysis of cytoplasmic O-PTIR spectra was used to identify microplastic-induced metabolic shifts. This chemometric analysis suggested that microplastic exposure resulted in oxidative stress and metabolic disruption, indicated by conformational protein changes-including early signs of misfolding and aggregation-which are hallmarks of a stress-adaptive cellular response. Our findings demonstrate that O-PTIR is an efficient, non-destructive technique for label-free microplastic studies in biological samples, enabling the identification, localization and characterization of heterogeneous microplastics, while simultaneously revealing their impact on cellular function and composition.