Introduction

Idiopathic Parkinson’s disease (iPD) is the most common movement disorder and a prevalent neurodegenerative condition in humans1. While aging is the primary risk factor, genetic and environmental factors - including pesticides, metal ions, and ionizing radiation (IR) also contribute to its etiology2,3,4. Neuroinflammation and oxidative stress also are strongly linked to iPD progression5,6.

High-dose radiation (HDR) exposure induces neuroinflammation, oxidative stress, and disruptions in DNA repair and cell survival mechanisms, potentially leading to progressive radiation-induced neurodegeneration7,8,9,10. HDR has been shown to impair hippocampal neurogenesis, contributing to long-term cognitive decline, particularly in younger individuals undergoing brain radiotherapy11,12,13. Epidemiological studies suggest that chronic HDR exposure may increase the risk of neurodegenerative diseases, including iPD, likely due to excessive oxidative stress and overproduction of reactive oxygen species (ROS)14,15.

Dopaminergic neurons in the substantia nigra (SN) are particularly vulnerable to oxidative damage, mitochondrial dysfunction, and calcium dysregulation - all key contributors to iPD pathogenesis16,17. Low-dose radiation (LDR), defined here as total γ-radiation exposure below 2 Gy (the maximum daily dose in standard brain tumor radiotherapy), may also impact mitochondrial function by damaging mitochondrial DNA (mtDNA) and increasing ROS production18,19. For clarity, while the UNSCEAR 201220 report defines “low dose” exposure for stochastic risk modeling purposes as ≤100 mGy, classifications for low-dose exposures differ based on the biological context. In radiation biology, particularly in preclinical and translational research, the term “low-dose” is often defined relative to therapeutic, sublethal, and deterministic thresholds. In our study, 1.79 Gy is considered low-to-moderate in the context of whole-body exposures in large animal models and relative to the fractionated doses (2–6 Gy/session) typically used in human cranial radiotherapy. Moreover, several studies have examined neurobiological and behavioral outcomes in rodent or NHP models using single-dose exposures between 1 and 3 Gy and referred to these as “low-dose” in the context of experimental radiation paradigms21,22,23. For the purposes of this study then, we refer to 1.79 Gy dose as an LDR dose that is, as a sublethal, subclinical exposure, in alignment with prior translational studies using similar dose paradigms in large animals22.

In general, animal studies suggest that chronic LDR induces oxidative stress markers, potentially triggering early iPD-related mitochondrial dysfunction24,25. However, most of these studies rely on rodent models, which differ significantly from large mammals such as primates (human and non-human) in metabolism, repair mechanisms, and radiosensitivity26,27, limiting then their direct translational applicability to humans.

At the molecular level, α-synuclein (α-syn) - a presynaptic protein abnormally accumulated in Lewy bodies (LB) and Lewy neurites (LN) - plays a central role in iPD pathology28,29. Oxidative modifications such as phosphorylation and truncation are linked to dopaminergic neuron degeneration30. Radiation-induced oxidative stress has been shown to promote α-syn aggregation, mitochondrial dysfunction, and endoplasmic reticulum stress, exacerbating neurodegeneration31. Other PD-associated proteins, such as Parkin, are similarly susceptible to stress-induced misfolding32. However, most studies exploring the link between radiation and iPD mechanisms rely on rodent models or in vitro systems, which may not fully capture the complexity of human iPD pathogenesis or the effects of γ-radiation on early disease events.

Given the lack of studies on the impact of γ-radiation on the SN in large mammals, investigating this relationship in swine or non-human primates may offer more relevant insights into the potential PD-related effects of ionizing radiation, particularly at low doses33,34. Unfortunately, such large-animal studies remain scarce.

To address this gap, we examined the effects of acute total-body low-dose γ-radiation (<2 Gy) on the SN of Göttingen minipigs, a large mammal model with strong translational relevance to humans35. Specifically, we assessed changes in the expression of key iPD markers, including α-syn, phosphorylated α-syn (pα-syn), tyrosine hydroxylase (TH), and PD-related genetic risk factors such as LRRK2, GBA, VPS13C, and Cathepsin D variants36. To evaluate neuroinflammatory responses, we measured glial fibrillary acidic protein (GFAP), a marker of astroglial activation. Additionally, mitochondrial alterations were evaluated through ATP5A, SDHB, and NDUF8 expression37,38. Lastly, we conducted histological and immunohistochemical (IHC) analyses of the SN, with a particular focus on dopaminergic neurons.

Dopaminergic neurons play a critical role in iPD pathogenesis and are essential for movement, learning, reward processing, and behavior across species39. Their degeneration underlies both motor and psychiatric symptoms in iPD40. Despite extensive research, the selective vulnerability of SN dopaminergic neurons, particularly in the pars compacta (SNpc), remains poorly understood. Hypotheses include dopamine toxicity, iron accumulation, and dysregulated pacemaking activity contributing to selective vulnerability in PD41,42.

This study aimed to determine whether LDR exposure (<2 Gy) induces molecular changes linked to iPD pathogenesis in a large animal model, providing novel insights into human responses to LDR exposure.

Results

WB analyses in the SN of RAD vs. SH group demonstrated no significant differences for all PD related markers examined: α-syn, p-α-syn, TH, GFAP, RAB12, GBA, VPS13C and CathepsinD (Fig. 1). Likewise, no significant differences were detected for all targeted mitochondrial markers: ATP5A, SDHB and NDUF8 (Fig. 2).

Fig. 1: Single exposure to total body radiation did not alter Parkinson’s related proteins in the substantia nigra.
figure 1

A Western blot densitometric analysis for PD related markers in the substantia nigra from the brains of Sham (n = 8) (blue) and Irradiated (n = 6) (green) Gottingen minipigs 33–35 days post total body radiation. B Representative western blots for all proteins are shown. Each blot was run in triplicate, and the graphs represent the average of 3 runs. * indicates p < 0.05 as determined by unpaired 2-tailed t-test. Error bars represent the standard error of the mean. Original full-length blots are presented in Supplementary Figs. 3–4.

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Fig. 2: Single exposure to total body radiation did not alter mitochondrial related proteins in the substantia nigra.
figure 2

A Western blot densitometric analysis for mitochondrial related markers in the substantia nigra from the brains of Sham (n = 8) (blue) and Irradiated (n = 6) (green) Gottingen minipigs 33–35 days post total body radiation. B Representative western blots for all proteins are shown. Each blot was run in triplicate, and the graphs represent the average of 3 runs. * indicates p < 0.05 as determined by unpaired 2-tailed t-test. Error bars represent the standard error of the mean. Original full-length blots are presented in Supplementary Fig. 5.

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Additionally, despite the multiple attempts and methodological approaches to detect and measure SN levels of lrrk2 protein in both SH and RAD group, we were not able to detect a WB signal of sufficient quality (based on the anti-lrrk2 antibodies commercially available) in order to precisely and consistently measure lrrk2 (see discussion about the levels of lrrk2 normally expressed in the SN).

The neuropathologic assessment of the SN in RAD vs. SH animals did not show any sign of neuronal morphometric alterations (Fig. 3, panels A, B, E, F with HE and CV stain, respectively). Also, IHC for TH stain showed an indistinguishable pattern of TH positivity between RAD and SH SN (Fig. 3, panels C and G). Finally, except for sporadic astroglial cells showing a mild level of reactivity, the GFAP stain did not show any marked difference in terms of astroglial response between the two groups analyzed (Fig. 3, panels D, H). To notice, the absence of neuromelanin is a normal condition in most mammalian species, including NHP, since it has been observed almost exclusively in humans43.

Fig. 3: Histological and immunohistochemical outcomes in the substantia nigra of Gottingen minipigs after total body radiation.
figure 3

The figure shows various histological and IHC stains for the SN of RAD and SH swine. HE stain (panels A and E) of the SN region in RAD vs. SH did not demonstrate any obvious difference between the two conditions. No vascular or perivascular lesions were observed. Also, to notice, the absence of neuromelanin pigment in the dopaminergic neurons of the swine SN. CV stain (panels B and F) did not evidence cellular or subcellular morphometric changes in RAD vs. SH SN cells, either neuronal cells or glial cells. The TH stain (panels C and G) clearly show positivity for tyrosine hydroxylase (TH) of the dopaminergic neurons of the swine SN. No difference in terms of IHC intensity between RAD vs. SH condition were detectable. GFAP stain (panels D and H) shows the absence of significant differences in terms of astroglial reactivity between RAD and SH in the SN of swine. IHC immunohistochemistry; HE hematoxylin and eosin; CV cresyl violet; TH tyrosine hydroxylase; GFAP glial fibrillary acidic protein; RAD irradiated animals; SH sham, non-irradiated animals; SN substantia nigra. The bottom left line underneath panel E. indicates the corresponding length (200 μm) of all images taken at the same level of magnification.

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Discussion

This study investigated the impact of acute single total-body low-dose radiation (LDR) exposure (1.79 Gy) on the substantia nigra (SN) of swine, a large mammal model with strong neuroanatomical and neurophysiological similarities to humans. Our findings indicate that a single acute total-body LDR exposure does not induce significant molecular, mitochondrial, or obvious histopathological changes in the SN within the four-week post-exposure period. The absence of alterations in key Parkinson’s disease (PD)-related markers, mitochondrial proteins, and histopathological features suggests that an acute subclinical radiation dose may not be sufficient to trigger early neurodegenerative processes in the SN.

WB quantitative analyses revealed no significant differences in the expression of α-synuclein (α-syn), phosphorylated α-syn (p-α-syn), tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and genetic PD-related markers (RAB12, GBA, VPS13C, Cathepsin D) between RAD and SH animals. Likewise, mitochondrial markers (ATP5A, SDHB, and NDUF8) remained unchanged, suggesting that an acute LDR exposure does not disrupt mitochondrial homeostasis in the SN in the short term. This is particularly relevant given that mitochondrial dysfunction and oxidative stress are key contributors to PD pathogenesis. Previous studies in rodent models have shown increased oxidative stress and mitochondrial damage following radiation exposure18,19, but our results suggest that such effects may not be as prominent in larger mammals after a single exposure to subclinical γ-radiation doses. More specifically, while rodent studies have reported oxidative stress, microglial activation, and cognitive impairments at doses ≤1 Gy22,44, the absence of similar effects in our Göttingen minipig model may reflect species-specific differences in radiation response, brain complexity, or regional susceptibility, further emphasizing the need for large animal validation.

Despite multiple methodological approaches, we were unable to detect a reliable LRRK2 protein signal in either RAD or SH animals. This is consistent with previous reports indicating that lrkk2 expression in the SN is relatively low in many mammalian species, including rodents and non-human primates (NHPs). LRRK2 mutations are among the most common genetic contributors to PD, yet its precise physiological role in the SN remains poorly understood. Some studies suggest that LRRK2 is more abundantly expressed in microglia and other non-neuronal cell types rather than in dopaminergic neurons45,46. Given the challenges in detecting endogenous LRRK2 in the SN, alternative methods such as RNA sequencing or mass spectrometry-based proteomics may be necessary for a more sensitive assessment of radiation-induced changes in LRRK2 expression in future studies.

Histopathological assessments further supported our molecular findings. Hematoxylin and eosin (HE) and Cresyl Violet (CV) staining revealed no neuronal loss, morphometric abnormalities, vascular or perivascular lesions in RAD vs. SH animals. Immunohistochemical (IHC) analysis of TH, a key enzyme in dopamine biosynthesis and a marker of dopaminergic neurons, showed a comparable distribution of TH-positive neurons between the two groups, indicating that LDR exposure did not compromise the structural integrity of the dopaminergic system at the histological level. Additionally, GFAP staining showed only mild sporadic astroglial reactivity without significant differences between RAD and SH groups, suggesting that LDR did not elicit a robust or sustained neuroinflammatory response. These results contrast with previous studies showing that high-dose radiation (HDR) exposure can induce pronounced neuroinflammation, particularly in the hippocampus7,11. The absence of substantial astrocytic activation in our LDR-exposed swine may indicate a threshold effect, where neuroinflammation becomes evident only at higher radiation doses or with prolonged exposure.

Species-specific differences in neuromelanin accumulation may also be an important factor in interpreting our findings. Swine, like most non-human mammals, do not develop significant neuromelanin accumulation in the SN even at older ages47,48. Neuromelanin is considered a key element in human SN neurons, where it plays a role in iron sequestration and oxidative stress modulation. This has led to the hypothesis that neuromelanin may contribute to the selective vulnerability of dopaminergic neurons in PD49. The absence of neuromelanin in swine SN neurons could partly explain why LDR did not induce oxidative stress-related neurodegenerative changes in this model. Furthermore, the lack of neuromelanin in the swine SN is a key species-specific limitation, as neuromelanin is known to bind iron and contribute to oxidative stress vulnerability in human SN neurons50. This may partially explain the absence of dopaminergic injury in our model despite a dose that would likely impact neuromelanin-rich neurons.

Although our study suggests that a single acute total-body LDR exposure does not induce immediate molecular or structural changes in the swine SN, several critical questions remain. First, our study did not address the effects of chronic or repeated LDR exposure. Epidemiological studies have suggested a possible association between chronic radiation exposure and an increased risk of neurodegenerative diseases, including PD14,15. Therefore, it remains to be determined whether cumulative LDR exposure over extended periods could lead to progressive PD-related alterations. Second, while our study focused on molecular and histopathological markers of early PD-related changes, functional assessments such as dopaminergic neurotransmission studies and behavioral analyses would provide further insight into potential subclinical effects of LDR on nigrostriatal function. Also, mechanistically, several factors may contribute to the lack of detectable pathology in the SN following whole-body exposure such as robust systemic and neuronal repair mechanisms, particularly in relatively radiation-resistant brain regions such as the SN, may have prevented sustained damage or facilitated rapid recovery; transient molecular or cellular perturbations may have occurred shortly after exposure but resolved by the 4-week endpoint (prior studies have demonstrated biphasic or time-dependent responses in radiation-sensitive tissues51; region-specific differences in radiosensitivity within the brain may result in differential vulnerability. While regions such as the hippocampus exhibit marked sensitivity to even moderate radiation doses, the SN may possess relative resistance under specific conditions. Additionally, we also acknowledge the distinction between whole-body and localized (e.g., cranial) exposures. Localized cranial irradiation often results in higher localized dose delivery with greater retention of biological effects in the targeted region (e.g., inflammation, oxidative stress, or demyelination), whereas whole-body exposure can lead to systemic counter-regulatory or adaptive responses (e.g., hematological or immune-mediated modulation) that mitigate CNS effects52,53. Future studies should incorporate in vivo imaging techniques, such as positron emission tomography (PET) or functional MRI, to assess alterations in dopaminergic activity following LDR exposure. Furthermore, while the 4-week endpoint was appropriate for identifying early indicators of injury or resilience, we recognize that this represents a limitation of the current study and that extended observation periods (e.g., 3–6 months or longer) are essential to fully characterize potential long-term or cumulative radiation effects, particularly in relation to Parkinson’s disease (PD)-relevant pathophysiology. Future studies will be designed to include longitudinal histopathological and behavioral evaluations at multiple timepoints to better define the temporal trajectory of radiation-induced changes in the SN and related basal ganglia structures.

Another important aspect to explore is the potential interaction of LDR with other environmental stressors, such as chronic inflammation, neurotoxic agents, or genetic predisposition. PD is thought to arise from a complex interplay of genetic and environmental factors, and it is possible that LDR may act as a sensitizing factor rather than a direct neurotoxic agent. More specifically, LDR has been shown to induce persistent reactive oxygen species (ROS) production and low-grade neuroinflammation, two mechanisms strongly implicated in the pathogenesis of Parkinson’s disease54,55. Additional mechanisms include: microglial priming, whereby irradiated microglia exhibit exaggerated responses upon subsequent inflammatory or toxic challenge21; mitochondrial dysfunction and oxidative DNA damage, which can synergize with known mitochondrial toxins (e.g., MPTP, paraquat); disruption of the blood–brain barrier (BBB), which may allow for increased permeability to circulating xenobiotics56; Epigenetic reprogramming or long-term changes in gene expression affecting stress and detoxification pathways. Actually, this framework theoretically aligns with the “two-hit” hypothesis, in which LDR constitutes the first, silent insult, rendering the brain more vulnerable to a second hit - such as pesticide exposure or dopaminergic neurotoxins - both of which are implicated in idiopathic Parkinson’s disease (iPD).

Investigating these interactions in future studies could provide a more comprehensive understanding of how radiation exposure influences neurodegenerative processes.

Lastly, given the critical role of mitochondrial homeostasis in PD pathogenesis, it will be important to assess the long-term effects of LDR on mitochondrial function and oxidative stress responses. While our study found no acute mitochondrial alterations, previous research suggests that radiation exposure can induce subtle but progressive mitochondrial impairments over time57. Longitudinal studies examining mitochondrial bioenergetics, oxidative DNA damage, and mitophagy in LDR-exposed models would help determine whether delayed neurodegenerative effects emerge beyond the short-term post-exposure window we investigated. Also, various studies have shown deleterious effects above 100 mGy in rodents, but most of them analyzed these effects at different endpoints or after protracted exposures. Our swine model, though, may have a different response threshold. Moreover, radiation effects are also related to dose-rate, species, brain-region, and timepoint of behavioral and pathological assessments. In this study, our focus was on the SN at 4 weeks, which may have delayed or region-specific vulnerability. Moreover, there are different studies not only supporting the hormetic effects of radiation but also the lack of neurodegeneration at similar doses21,44. These last considerations highly support an urgent expansion of dose-effect, timing and brain region-based analyses in large animals.

Among the limitations of our study there is the absence of behavioral or functional assessment. Indeed, while behavioral or functional testing (e.g., motor performance, PET imaging of dopaminergic tone) would have enhanced translational interpretations and potentialities, these assessments were beyond the primary scope of this acute, observational study. However, future work will need to incorporate longitudinal behavioral and neuroimaging endpoints to assess functional consequences of LDR in these large animals.

Also, in terms of neuropathological assessment the absence of overt pathology may reflect biological resilience or the limitations of our quantification approach, which did not include, among other, unbiased-stereology quantifications. In fact, we cannot totally exclude that subtle or spatially restricted changes may thus have gone undetected. Further neuropathological analyses, including unbiased-stereology based assessments should be performed in the future.

Importantly, while a formal dose conversion between species was not performed using specific allometric scaling, we based our parameters on published data from NASA and radiation biology studies58,59, where 1.79 Gy whole-body in non-human primates (NHPs) is broadly equivalent to moderate acute exposures in humans as seen in certain occupational, accidental, or spaceflight contexts.

Here, it would be important to describe possible realistic human scenarios in which individuals might be or have been exposed to radiation doses comparable to the 1.79 Gy as used in our study. These are some of the possible human scenarios:

Radiological accidents

Acute doses in the range of 1–2 Gy have been reported in historical nuclear accidents such as the Chernobyl disaster (1986) and the Tokaimura criticality accident (1999). In these events, some first responders and workers experienced whole-body doses within the 1–2 Gy range, which were below lethal thresholds but associated with subclinical or early prodromal symptoms60,61.

Space missions

Astronauts, particularly during solar particle events (SPEs) or extended deep space missions (e.g., to Mars), may receive cumulative or acute doses approaching or exceeding 1 Gy62. Although current shielding strategies mitigate risk, peak exposures during unshielded events remain a concern.

Radiation therapy accidents

While therapeutic radiation typically involves localized exposure, rare radiation therapy accidents or planning errors have resulted in unintended whole-body or large-field doses in the sub-lethal range (~1–2 Gy), occasionally causing systemic symptoms or cognitive complaints63.

Diagnostic radiation cumulative exposures

In rare cases of extensive medical imaging, cumulative doses may approach this threshold. For example, multiple full-body CT scans (each delivering ~10–30 mGy) would require over 50–100 scans to approach 1.79 Gy cumulatively. Though this is clinically unusual, such cumulative exposures may occur in patients with chronic health surveillance requiring repeated imaging over years64.

Our study provides novel insights into the effects of acute LDR exposure on the SN in a large mammalian model. Our findings suggest that a single subclinical dose of γ-radiation does not induce short-term molecular or structural alterations associated with PD in the swine SN. However, given the complex interplay between radiation exposure, oxidative stress, and neurodegeneration, further research is warranted to assess the long-term consequences of LDR, particularly in the context of chronic or repeated exposures. By leveraging large animal models such as swine, future studies may provide valuable translational insights into the potential role of radiation exposure in PD pathogenesis and other neurodegenerative disorders.

Methods

All methods and procedures used for this investigation have been described earlier65. Here, we provide a brief description of the animal use and methods relevant for this specific substantia nigra (SN) focused investigation.

Animal procedures

All animal handling procedures followed the guidelines of the National Research Council for the ethical handling of laboratory animals and was approved by the Uniformed Services University (USU, Bethesda, MD) and Armed Forces Radiobiology Research Institute (AFRRI, Bethesda, MD) Institutional Animal Care and Use Committees (Protocol PHA-18-942) in compliance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animals, and all applicable Federal regulations governing the protection of animals in research. Male Göttingen minipigs ranging in age from ~5.0 to 6.5 months were purchased from Marshall Farms Group Ltd (North Rose, NY, USA). Animals were pair housed in a barrier facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). Housing rooms were maintained at 21 ± 2 °C, 50 ± 10% humidity, and 12-h light/dark cycle with food and water available, ad libitum. Swine (minipigs) were divided into the following two groups:

  1. 1.

    Radiation (RAD) (n = 6)

  2. 2.

    Sham (SH) (n = 8)

Approximately two weeks after arrival, minipigs receiving total-body radiation were deeply anesthetized with Telazol/Xylazine (4.4 mg/kg–2 mg/kg) and transported to the High-Level Cobalt facility at AFRRI. Animals were exposed one at a time, bilaterally, to a target total body dose of 1.79 Gy of Cobalt (60Co) radiation delivered at a dose rate of 0.485–0.502 Gy/min65. After radiation procedures, each animal was transported back to the housing facility for recovery. Minipigs assigned to the sham (SH) groups were also deeply anesthetized with the same dose of Telazol/Xylazine in the housing facility but were not transported to the Cobalt facility.

The total number of SH and RAD animals listed above are a combination of Vehicle-treated (Veh) or Captopril-treated (Cap) animals from a larger cohort of animals66. Previously, we have demonstrated that there were no statistical differences between SH- Veh and SH-Captopril animals or between RAD-Vehicle and RAD-Captopril animals in brain related outcomes65. Again, in all cases represented in this study, there was no statistical difference between the Veh and Cap groups for all molecules targeted (Supplementary Figs. 1–2). Thus, the Veh and Cap animals were combined for each respective SH and RAD group resulting in the following total number of animals per group: n = 8 for SH and n = 6 for RAD.

Euthanasia and tissue collection

30-days after total body radiation, euthanasia was performed with an intracardial injection of Euthasol (4.5 ml/kg) and confirmed by lack of heartbeat. Each animal underwent necropsy procedures for the sampling of different organs as well as the collection of the entire brain and spinal cord. All animals were sacrificed 4 weeks (±2 days) post exposure.

Each brain was grossly inspected and longitudinally dissected across the median line of the corpus callosum to separate the two cerebral and cerebellar hemispheres. The left hemisphere of each animal was quick-frozen in chilled liquid isopentane on dry ice (for molecular analyses). Frozen brains were kept at –80 °C until use. The right hemispheres were placed in 10% buffered formalin for tissue fixation (for histological and immunohistochemistry purposes).

Protein extraction and Western blot (WB) procedures

Each left cerebral hemisphere was cut into 100 µm thick sections in a cryostat and further microdissected by anatomical regions, including but not limited to Substantia Nigra (SN). The dissections were guided by following the Gottingen Minipig Brain Atlas (https://cense.au.dk/fileadmin/ingen_mappe_valgt/fileadmin/minipigatlas/atlas/index.html).

Tissue lysis, total protein quantifications, and WB procedures were carried out as previously described65. All WBs were run in triplicate and averaged for each antibody with all protein signal intensities normalized to total protein content as determined using No-Stain Protein Labeling Reagent (A44717, Invitrogen, Carlsbad, CA). Blots were imaged on the iBright FL 1500 Imaging System (A44241, Invitrogen, Carlsbad, CA) and densitometry was performed with IBright Analysis software (Version 5.3.0, Life Technologies-Thermo Fisher Scientific, Waltham, MA).

Primary antibodies for Western blot

The following primary antibodies were used: tyrosine hydroxylase (TH), a tyrosine 3-monooxygenase enzyme catalyzing the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) (1:1000, MAB318, Millipore, Billerica, MA); alpha synuclein (α-syn), a protein codified by the SNCA gene regulating the synaptic vesicle trafficking and neurotransmitter release (1:5000, ab138501, abcam, Waltham, MA); phospho-alpha-synuclein (pα-syn-S129) (1:250, GTX50222, Genetex, Irvine, CA); Rab12, a member of the small GTPase superfamily (1:1000, 18843-1-AP, Proteintech, Rosemont, IL); Cathepsin D, a lysosomal protease that is ubiquitously expressed and has been shown to be involved in the pathogenesis of several diseases, including PD (1:2500, 21327-1-AP, Proteintech, Rosemont, IL); GBA1, a lysosomal protein which when mutated has been shown to be a genetic risk factor for PD, (1:500, 2792-1-AP, Proteintech, Rosemont, IL); VPS13C, a vacuolar protein associated with loss of function mutations related to early onset PD, (1:2000, 29844-1-AP, Proteintech, Rosemont, IL); GFAP, an astrocytic marker (1:10000 NCL-L-GFAP-GA5, Leica Biosystems, Deer Park, IL); ATP5A, an ATP synthase widely expressed in the inner mitochondrial membrane essential for cellular energy production (1:1000, ab14748, abcam, Waltham, MA); SDHB, also known as Complex II, located in the mitochondrial inner membrane essential for energy metabolism and complex cellular processes (1:500, ab14714, abcam, Waltham, MA) and NDUF8, a subunit of the mitochondrial Complex I plays an essential role in the electron transport chain and oxidative phosphorylation within the mitochondria (1:500, ab110242, abcam, Waltham, MA).

Secondary antibodies for Western blot

The following HRP tagged secondary antibodies were used: goat anti-mouse (1:2000, ab97040, abcam, Waltham, MA and goat anti-rabbit (1:2000, ab97080, abcam, Waltham, MA).

Neurohistological and immunohistochemistry procedures

Tissue blocks from each animal were uniformly processed using an automated tissue processor (ASP 6025, Leica Biosystems, Nussloch, Germany). After tissue processing, each tissue block was embedded in paraffin and cut in a series of 20 5µm-thick consecutive sections. The first two sections were respectively selected for hematoxylin and eosin (H&E), and Cresyl Violet (CV) stains, while the remaining sections were available for immunohistochemistry procedures. Sections encompassing the substantia nigra region from a total of 4 animals, 2 per treatment condition, were selected. 3 slides per animal were used. Immunohistochemistry procedures for anti-glial fibrillary acidic protein (GFAP) (1:250, mouse monoclonal antibody, Leica Biosystems, Buffalo Grove, IL) with bond heat-induced epitope retrieval, epitope retrieval time 10 min, PA0026, Leica Biosystems, Buffalo Grove, IL) was performed using a Leica Bond III automated immunostainer with a diaminobenzidine chromogen detection system (DS9800, Leica Biosystems, Buffalo Grove, IL). Immunohistochemistry procedures for tyrosine hydroxylase (TH) (1:500, mouse monoclonal, MAB318, Millipore-Sigma, Billerica, MA) were performed as follows. Slides were deparaffinized by placing at 60 °C for 30 min followed by xylene rinse, 3 ×10 min, and rehydration in a decreasing alcohol series (100%, 95% 70% EtOH), 2 ×10 min each, and a final rinse in distilled water. Heat induced antigen retrieval was performed by placing slides in 1% Citrate Buffer (pH6) and microwaved for 5 min followed by a slow return to room temperature for 45 min. Endogenous peroxidases were blocked using 0.3% H2O2 for 15 min at room temperature followed by a wash in 1X PBS, 3 ×10 min. Slides were blocked in 1X PBS + 4% goat serum +0.1% Triton X-100 for 1 h at room temperature followed by 1X PBS rinse, 3 ×10 min. Sections were incubated with TH primary antibody (1:500) diluted in 1X PBS + 3% goat serum overnight at 4 °C. Slides were rinsed in 1X PBS, 3 ×10 min and incubated with goat anti mouse biotinylated secondary antibody (1:500) (BA-5200, Vector Laboratories, Newark, CA) for 2 h at room temperature. Slides were rinsed in 1X PBS, 3 ×10 min followed by ABC peroxidase reagent (PK-6100, Vector Laboratory, Newark, CA) for 30 min at room temperature followed by rinse in 1X PBS 3 ×10 min. Finally, slides were developed using DAB chromogenic stain (D5905, Sigma, St. Louis, MO) and coverslipped with Eukitt mounting medium (15320, Electron Microscopy Sciences, Hatfield, PA).

All stained sections were scanned by an Aperio scanner system (Aperio AT2 - High Volume, Digital whole slide scanning scanner, Leica Biosystems, Inc., Richmond, IL) and stored in Biolucida system, a hub for 2D and 3D image data (version 2017, MBF Bioscience, Williston, VT, USA) for further assessment and analyses to verify the immunoreactivity (IR) for each antibody and histological distribution of possible lesions and their severity across the examined brain region and conditions. A preliminary neuropathologic assessment for each section, region and animal was performed using Aperio ImageScope (Aperio ImageScope, version 2016, Leica Biosystems, Inc.). After a preliminary ImageScope inspection (max 20X), a Zeiss Imager A2 (ImagerA2 microscope, Zeiss, Munich, Germany) bright-field microscope inspection at higher magnification (40X, 63X oil-immersion objectives) was used to identify and digitally photograph possible histopathologic details as needed.

Statistics

The number of animals used in this study (SH = 8; RAD = 6) represented a portion of a much larger swine cohort that was part of a multiyear investigation of LDR effects across different tissues. Preliminary power analyses from previous investigations consistently showed that the sample size used in this study, or even smaller as in our previous investigation, was sufficient to obtain significant results considering the variability of the measurements performed in these studies. For each antibody and each examined region, two-tailed unpaired t-tests were performed for WB. Data values reported are mean ± standard error of the mean (SEM). Differences with p-value ≤ 0.05 were considered significant in all cases. Statistical tests were performed using GraphPad Prism version 9.0.2 for Windows (GraphPad Software, La Jolla, CA).