Animal usage

Male Sprague Dawley (SD) rats, 9–10 weeks of age, were procured from Charles River and were handled in accordance with the VA New Jersey Health Care System Institutional Animal Care and Use Committee (IACUC). Rats were housed individually in standard polycarbonate 18 quarts tubs with bed-o-cob bedding which was changed once a week. Individual housing was necessary due to planned behavioral experiments, and the unknown effect of r-mTBI together with PFF on the interaction between injured males in the same cage. Rooms were kept at 22 °C +/− 4 °C and rats were kept 12 h on and 12 h off reverse light cycles to allow for behavioral experiments during rat subject active phase. To minimize stress during handling and behavioral experiments rats were acclimated to the experimenter by handling for a week before experiments were performed. All behavioral experiments were performed under red light. Three separate cohorts were used in this study. Cohort 1: 4× r-mTBI and shams for behavior and histology, cohort 2: 8× r-mTBI and shams for behavior and histology, cohort 3: nucleation of PD by injection of PFFs followed by 8× r-mTBI for histology.

Recombinant αSyn PFF synthesis

PFFs were generated in the lab of Dr. Andrew B. West at Duke University School of Medicine as previously described by Abdelmotilib et al. [26], with purification and quality control performed using dynamic light scattering (Fig. 5S). Briefly, murine αSyn was expressed in E. Coli, and produced in large batches. Crude protein extraction was achieved followed by size exclusion chromatography and ion exchange. Endotoxin was also removed. Purified αSyn was then aggregated into higher order fibrils using an aggregation assay by shaking at 37 °C for a week in buffer [24, 32]. These fibrils were fractured in a sonicating water bath, diluted to 5 µg/ml, and injected in a 4 µl volume over 30 min for total of 20 µg per injection site.

Intracranial injection of monomeric αSyn, PFFs, or LPS

Rats were injected with monomeric αSyn to serve both as a vehicle and surgery control. PFF injection was used to corrupt the endogenous αSyn and promote human PD-like inclusion formation and pathology. Lipopolysaccharide (LPS) injection was used to validate antibodies and detection of astroglia expansion and microgliosis. Rats were anesthetized with 5% v/v isoflurane with a flowrate of 0.8 L/min. Once fully anesthetized, the rat fur was clipped, and the incision area cleaned with an alcohol wipe, and eye ointment was also applied. The rat was then placed on a heating pad in prone position, secured in the stereotaxic surgical system with ear bars, and kept under with a canulae delivering continuous O2 flow at 0.8 L/min and isoflurane setting of 2.5% v/v. Prior to incision the surgical area was further cleaned with chlorohexidine. For analgesia, subdermal injection of 0.1 ml Marcaine at 2.25 mg/kg was injected at each end of the planned sagittal incision which was made down the midline from approximately the frontal bone, moving caudally to the base of the skull to expose the entire calvarium. Once the incision has been made, the wound area was irrigated with sterile saline, and fascia attached to the skull was gently removed with sterile cotton applicators. To facilitate exposure of bregma, 3% hydrogen peroxide solution was applied directly to the skull. Once the bregma was clearly visible, a burr hole was made at the coordinates: X = 2.5, Y = 5.35–5.5, Z = 7.4–7.5 mm of Bregma. 4 µl volume of a 5 µg/µl PFF solution, monomeric alpha synuclein, or Lipopolysaccharide (LPS) was injected over 30 min. The needle was left in the injection site for 5 min after dispensing to prevent reflux along the injection track. The needle was retracted very slowly. Contralateral (left) side was intentionally left un-injected to serve as an internal control. After injections were complete, the surgical area of the exposed calvarium was irrigated with sterile saline, and the incision was closed with nylon surgical sutures. The sutured area was cleaned once more with 3% hydrogen peroxide and the rat was placed in a recovery cage with food and water on a heated pad. The rat was monitored for seizures and signs of recovery. After the initial 1-h observation during recovery from anesthesia the animal was placed back in the home cage, monitored once every hour, and then once every four hours.

Traumatic brain injury

The injury device was designed to approximate the most common type of head injuries which involve an impact to a large part of the skull from a fall, strike to the head, or contact with a hard surface during a vehicle collision. The injuries were performed once every 14 days to accommodate behavioral tests and every 10 days starting at 1 month after injection of PFFs and/or monomeric αSyn. Fully anesthetized male SD rats, 8–10 weeks of age, were placed on ∼10 cm, 2.84 N/cm stiffness foam platform in a prone position to allow for dorsoventral and anteroposterior acceleration and deceleration of the head [29] under a 2.5 cm diameter suspended guide tube covering the unshielded calvarium (Fig. 1A). Stainless steel injury weights ranging from 0.250 to 2.0 kg with 0.250 kg increments were loaded 25 cm above the head into the guide tube and held in place with a pin. The injury was initiated by rapidly pulling the pin allowing the weights to free fall 25 cm down a guide tube impacting the head. The weights were immediately retracted up the guide tube using an attached string. Injuries were titrated and TBI was defined as the highest survivable single injury (1.750 kg released from 25 cm and calculated impact of 4.29 J) within a 10–14-day period, not resulting in skull fracture or damage to the skin. We define mTBI (1.500 kg released from 25 cm with calculated impact of 3.68 J) as the highest survivable repetitive injury.

Fig. 1

Gross pathology indicating encephalomalacia in rat brain following 8× rmTBI. Novel surgery-free, unshielded weight drop injury device designed to impact the entire calvarium similar to a fall, car accident, or strike to the head (A). Titration of injury severity 0–2.0 kg with 0.250 kg increments indicating instant fatality following a single 2.0 kg injury, instant fatality following second injury with 1.75 kg and survivability of 4 injuries with 1.5 kg weight drop injury from 25 cm height. B Behavioral assessment of motor deficits using an open field test 10 days post injury indicating no behavioral deficit after up to 8× r-mTBI. C Fluorescent staining for NeuN+, Iba1, MHCII, GFAP of an area of encephalomalacia (D1–D10). NeuN+ cells are absent from the area of encephalomalacia (D1–D2). The area of injury devoid of neurons (NeunN+) is infiltrated by Iba1+ and MHCII + microglia (D2–D4). Expansion of GFAP+ astrocytes in the area of injury (D10). An area of brain thinning or encephalomalacia is present and visible on the anatomical left cortex on an intact brain prior to sectioning. Dashed white line and square indicate region that was magnified in D2–D5 and D7–D9

Open field test for assessment of ambulatory activity

On day 1, starting with the open field, behavioral tests were performed on rats that underwent 8× r-mTBIs and cohort sham controls in the second week after each of the 8 injuries to measure long lasting consequences of r-mTBI. On day 1, rats were placed individually in a 72.4 cm by 72.4 cm square open arena, for 5 min with the center zone set 10 cm away from the edge of the arena. Area of the center zone is equal to that of the perimeter zone (area between wall of the arena and perimeter of the center zone). ANY-maze software version 6.17 was used to track the rat speed and location through a ceiling mounted camera.

Novel object recognition and novel object placement memory tests

On day 2 of behavior, two identical objects were placed equidistant from each other, and the walls of the arena allowing animals to acclimate and explore the objects for 5 min. On day three (memory), two new objects were placed inside the arena; the rat was placed inside along the wall at the start of the test and allowed 5 min to explore. The animals were then placed back on the cart and remained in the test room for four hours after which one of the objects was replaced with a new object to test for novel object recognition. Position of the new object was alternated between left and right location to account for potential area rather than novel object preference. Again, animal position was tracked, and time spent exploring objects was recorded using Anymaze 6.17 software. On day four, similar to day 3, animals were placed into the arena with two new objects for five minutes (learning), and after a four-hour delay one of the objects was moved to a new location. Time spent exploring the objects at the original and new location was measured. Initial object selection criteria were made on the basis that the object cannot be moved by the rat, cannot be climbed on, can be easily cleaned, does not emit an odor, and had a variable textured surface. All objects were calibrated with non-experimental naïve rats prior to use to avoid aversion or preference to a specific object rather than a novel object. Novel objects that were preferred by naïve animals, or toward which the animal showed aversion were excluded from use in the experiments. Objects used included the following types: soda can, glass vase, various plastic soda bottles, jars, and cups.

Perfusions and brain collection

Anesthesia was induced with 5% v/v isoflurane with a flowrate of 1.5 L/min. Once fully anesthetized, the rat fur was clipped, and the incision area cleaned with an alcohol wipe. The rat was then placed on a dissecting tray on its back, secured in place using pins, and kept anesthetized with a canulae delivering continuous O2 flow at 1.5 L/min and isoflurane setting of 5.0% v/v isoflurane. To ensure that the animal was fully anesthetized, hind limb pinch was performed to check for reflex. An incision was made, either on the right or the left lower quadrant below the rib cage, by cutting into the abdominal cavity. The incision was expanded along the one side by cutting the rib cage up to the clavicle, followed by an identical contralateral incision. The ribcage was then lifted and reflected, and the diaphragm was severed. The right atrium was cut for exsanguination, and a needle was inserted into the left ventricle, to first deliver 100 ml of PBS, followed by 100 ml of 4% PFA for fixation. The skull was opened using bone rongeurs. The exposed brain was removed gently using a small spatula and transferred to a 50 ml tube, with 4% PFA in PBS on a tumbler at 4 °C overnight followed by 48 h in 30% sucrose for cryopreservation. Lastly, the brain was rapidly frozen by submerging the cryoprotected brain for 2 min into methyl butane cooled with dry ice to − 40 to − 50 °C and stored at − 80 °C.

Immunohistochemistry

40-micron serial coronal sections of the frozen cortex and midbrain were obtained using a freezing microtome. A needle borehole was used to indicate the side contralateral to the injection of fibrilized or monomeric αSyn. SNpc containing brain regions were sampled every 4th section and the rest of the midbrain including striatum was sampled every 6th section. Sections were stored at − 20 °C in 50% glycerol with 0.1% sodium azide. Entire nigral and striatal representative samples to be stained were removed from cryopreservation media and washed in TBS. Sections were then exposed to antigen retrieval solution (10 mM sodium citrate, 0.05% Tx-100, pH 6.0). Antigen retrieval solution was washed from the free-floating section with TBS before blocking in 5% goat or donkey serum (depending on the secondary antibody species) and 0.3% Tx-100 in TBS. Sections were then incubated in primary antibody for 24 h and secondary antibody solutions for 2 h at 4 °C with agitation (Table 1). Once mounted on a slide the sections were counterstained with Hoechst 33342 for nuclei. Sections for confocal microscopy were wet mounted using Pro-Long Gold and covered before imaging. LI-COR IRDye secondary antibodies were used for visualization using the LI-COR Odyssey scanner (Table 1).

Table 1 Primary and secondary antibodies used in the studyFluorescent confocal microscopy and imaging

Sections were imaged in four channels using Olympus FluoView 3000 confocal microscope and Fluoview FV31S-SW software. Excitation wavelengths of 405 nm, 488 nm, 561 nm, and 640 nm were optimized in each channel before images were acquired. For counting TH, pSyn, and pTau, 10× magnification z-stack images (10 µm step size) of ipsilateral and contralateral sides were obtained. Each side was stitched together separately using the FV31S-SW software. Z-stack images for visualization of pSyn, pTau, and inflammatory marker pathology were obtained at 20× and 60× magnification using the same optimized laser settings. All images were exported and processed to MaxZ projections using ImageJ 1.53a [33].

Counting TH + neurons, Lewy body-like inclusions and pTau aggregates

PD related pathology was counted in the SNpc containing brain slices sampled every 4th section. 10× magnification z-stack images of SN stained for tyrosine hydroxylase (TH), phosphor-Serine-129 alpha synuclein (pSyn), and phosphorylated Tau (pTau) were processed in ImageJ to MaxZ projections. TH stain locality was used to identify and draw a region of interest (ROI) around the SNpc in each image. Files with drawn ROI were randomized using a better finder rename 10 software. The files were then assigned to be counted by a technician blinded to treatment groups and brain injection location for unbiased counting of dopaminergic neurons (TH+), Lewy bodies (pSyn+), and pTau+ inclusions. DNs were counted only if an entire cell body was within the drawn ROI that had TH+ staining. Axonal projections and non-spherical/partial cell bodies were not counted. Lewy body-like inclusions were counted with stringent exclusion criteria. Initial counts of Lewy bodies were obtained by identifying pSyn+ aggregates with stain intensity greater than 3× background intensity and co-localized to a TH+ neuron body. The pSyn+ inclusions had to be perinuclear and within the vicinity of other Lewy Bodies or Lewy neurites in the same or adjacent TH+ neuron. Images that met some but not all exclusion criteria were flagged and imaged at higher magnification. pTau inclusions were counted with several stringent exclusion criteria. A pTau+ aggregate was counted as an inclusion only if the pTau+ aggregate co-localized to a TH+ cell body and cell-like morphology was apparent. Punctate staining, i.e. 1–2 pixels of increased stain intensity, were not counted as a pTau inclusion. pTau inclusions were counted as total pTau inclusions per image as well as pTau inclusions that co-localized with pSyn+ Lewy bodies. Using the criteria each object was selected using multi point tool in imageJ, to click and record each counted object. In each case, the counts were normalized to the area of the SNpc reported in mm2. Samples of counting and selections are included in the supplemental section (Fig. 6S).

LICOR imaging

A LI-COR Odyssey CLx scanner was used to visualize gross brain pathology on coronal sections from 8× r-mTBI rat brains. Sections mounted on slides were scanned tissue side down without a coverslip on the scanner bed, with an offset of 0.00 mm and a 21 μm resolution. The area of capture was set using LI-COR software. Double staining with antibodies against Iba1 and MCHII (Table 1) were used to distinguish areas of authentic pathology from potential tissue damage during cutting and mounting. George Paxinos and Charles Watson SCR_017124 Rat Brain Atlas was used for reference [34].

s100Beta measure in blood for TBI ELISA and western

s100Beta, an astrocytic protein, has been reported as a peripheral blood biomarker of TBI severity in humans and rats [35]. The greatest blood brain barrier (BBB) breach was reported 2 h after a TBI in rats [36]. To determine the degree of BBB permeability caused by our TBI and mTBI, blood samples were collected from control and TBI rats 2 h after a closed head weight drop injury. Deeply anesthetized rats underwent a bilateral thoracotomy followed by a cardiac puncture for blood collection. Serum was separated out by centrifugation. Serum sample and quality controls were prepared per s100beta kit (BioVendor R&D®). instructions. Absorbance was measured using a plate reader at 450 nm, with the reference wavelength set to 630 nm.

For western blot analysis, 30 μg of serum protein was loaded on a 4–20% stain free Mini-Protein TGX Gel (Bio-Rad) for 30–40 min at 200 V. The gel was transferred to a methanol activated PVDF membrane, using Trans-Blot Turbo mini packs (Bio-Rad) on a Trans-Blot Turbo ™ System. After 7-min transfer, the membrane was washed and blocked using 5% blocking buffer in TBST. The membrane was probed with 3 most frequently cited and commercially available s100beta antibodies with corresponding HRP conjugated secondary (Table 1). Membranes were incubated with primary antibody in 5% blocking solution with agitation overnight at 4 °C. After washing, the membranes were then incubated for 2 h at 4 °C with appropriate HRP conjugated secondary and visualized using ChemiDoc MP imaging system (Bio-Rad). The signal was normalized to total protein on the membrane. Briefly, stain free gels allow for cross linking of loaded protein using UV light before transfer. After transfer UV is used again on the membrane, which allows for visualization of total protein bands on the membrane. All band quantification was performed using Image Lab studio for MacOS Version 6.1.0 build 7 2020. Dual Color Standard protein ladder was used for band size estimation. All transfer buffers used were obtained and used as indicated by the manufacturer’s instructions (Bio-Rad).

Statistics

Required sample size was determined experimentally, starting with n = 6 per group and increasing to n = 10 per group after initial fatalities and noted variability with behavioral experiments. For TH+ neurons zero values were not expected, ordinary one-way ANOVA was performed to test for significance with Holm-Šídák’s multiple comparisons test comparing all means to 8× r-mTBI+ monomer group. In counts of αSyn pathology where zero values were expected, the Kruskal–Wallis test was performed. Grubbs outlier tests were performed with alpha 0.01. In all groups where outliers were identified and removed from comparison, outlier values are included in the figure caption. Significance was indicated by *p < 0.05 and **p < 0.01. Open bar graphs indicate means, closed circles represent individual data points, and error bars represent standard error of the mean. Data was analyzed and graphed using Prism 9.3.1 for macOS.