Regulatory approval and participants
All study activities were conducted in accordance with University of Florida (UF) Gainesville Health Science Center Institutional Review Board (IRB-01) issued approval (IRB202300741). Study procedures, risks, benefits, and alternatives were explained to all participants, and informed consent was obtained according to IRB protocol after being provided written documentation of study participation. A waiver for written consent was allowed given that the only record linking the subject and the research would be the consent document and the principal risk would be potential harm resulting from a breach of confidentiality. All protocols were followed in accordance with UF IRB policies and regulations to conduct safe research with human participants.
This study on the feasibility of collection and analysis of residual brain tissue on deep brain stimulation (DBS) surgical instruments, particularly cannulas and microelectrodes, was conducted with de-identified samples without any HIPPA identifiers. Surgical instruments from DBS procedures performed by two functional neurosurgeons for patients with Parkinson's disease (PD) or essential tremor (ET) conducted at UF over a 6-month period were collected. Inclusion criteria were: patients between the ages of 18 and 89 years old undergoing a DBS procedure performed for a pre-operative diagnosis of PD or ET. Diagnosis was made or confirmed by movement disorder neurologists at the University of Florida Norman Fixel Institute for Neurological Diseases. Selected patients were recommended for treatment with DBS by a multidisciplinary board of clinicians as previously described for both PD and ET 21,22. Included subjects underwent staged bilateral DBS lead implantation into either the ventral intermediate thalamic nucleus (VIM) for patients with ET, or the subthalamic nucleus (STN), globus pallidus internus (GPI), or VIM for patients with PD. Surgical instruments from a procedure were excluded if the designated investigator performing data analysis was a surgeon in the DBS procedure, if surgical instruments were contaminated during the procedure (for example, inadvertently dropped on the floor), or if surgical instruments were visibly bloody. The only demographic data collected from patients were their pre-operative diagnosis and surgical target for the DBS procedure they underwent (Table 1).
Surgical details and collection of surgical instruments
DBS target selection and surgical protocol was performed as previously described 23. After discussion of risks, benefits, and alternatives of the procedure, surgical consent was obtained. Patients underwent placement and registration of a stereotactic head ring for DBS lead targeting. All planned surgical trajectories traversed the posterior region of the frontal lobe anterior to the primary motor cortex, typically through the superior or middle frontal gyrus and subsequently through the corona radiata and deep white matter until the VIM, GPI, or STN was encountered as planned. A high-resolution contrasted MRI sequence was used to plan trajectories with care to avoid the ventricles, cortical vessels, and deep parenchymal vessels. After trajectory planning, patients were brought to the operative suite where the scalp was prepared and draped in the usual sterile fashion. Local anesthesia was utilized, and patients remained awake for the duration of the procedure. The stereotactic trajectory was marked, scalp and periosteal tissue divided in a linear incision, and a burr hole made with a high-speed drill. Hemostasis was obtained with bipolar electrocautery, and the dura mater overlying the target brain was cauterized and incised. A small entry point through the leptomeninges was created, and the standard 1.4mm diameter guide cannula with stylet (Alpha Omega, Nazareth, Israel) introduced into the parenchyma to a variable depth of ~ 3 to 6 cm depending on target. The inner stylet of the cannula was removed, and a shielded “NeuroProbe” electrode (Alpha Omega, Nazareth, Israel) introduced through the cannula and further into the parenchyma an additional 2.5 cm during microelectrode recordings. Intra-operative microelectrode recordings were performed by the neurology team to define the target region, optimizing lead placement. Once concluded, the microelectrode was removed and the cannula was further advanced 2.5 cm to the target structure (VIM, GPI or STN). The permanent DBS lead was implanted with further macrostimulation testing, and then the lead was secured in place and cannula removed. The cannula and microelectrode were placed into their original sterile packaging by members of the surgical team using sterile technique and placed in storage at 4°C until residual tissue purification from these devices was performed within 6 hours of collection as described below.
Purification of residual tissue from cannulas and electrodes
Cannulas and microelectrodes were transferred on ice in sterile packaging from the operative suite to laboratory facilities for purification of tissue. For each subject, the cannula (including stylet) and microelectrode were first inspected and the approximate portion of each which had been placed intra-parenchymal were marked and cut from the remaining portion with wire cutters. The intraparenchymal portions of the cannula and microelectrode were then further sectioned into ~ 1 cm portions and placed into a 1.5 mL microcentrifuge tube containing 150 µL of extraction buffer (0.2% sodium dodecyl sulfate (SDS), 1% Triton X-100 in 1x phosphate buffered saline (PBS), pH 7.4) at 25°C. Samples were then vigorously shaken on a vortex mixer for 5 minutes and then heated at 95°C for 5 minutes to solubilize and denature residual tissue on the cannula and microelectrode sections; this procedure was repeated three times to ensure maximal extraction of tissue. Samples were then centrifuged at 21,000 x g for 10 minutes at 25°C. Cannula and microelectrode fragments were removed from the microcentrifuge tube and placed into a miniprep column (filter removed; Promega) that was then attached to the top of the microcentrifuge tube; the microcentrifuge tube with a column in place was further centrifuged at 1,500 x g for 2 minutes at 25°C to drive residual liquid sample from fragments into the sample solution before discarding (Fig. 1). The sample was then stored at -80°C until subsequent analysis. Concentrations of samples were determined using the bicinchoninic acid (BCA) assay (Pierce, Waltham, MA) with bovine serum albumin (BSA) as the standard.
Retrieval and preparation of reference autopsy tissue
Human brain tissue was obtained through the University of Florida Neuromedicine Human Brain Tissue Bank in accordance with institutional review board approval. Post-mortem pathological staging and diagnoses were made according to respective neuropathological criteria for Lewy body dementia (LBD) 24. Unfixed samples of cingulate cortex from two post-mortem cases of LBD were retrieved and homogenized in 500 µL of extraction buffer detailed above. Case 1 was age 68 at death and case 2 was age 83 years at death, both with Braak stage VI of LBD pathology. Samples were then similarly shaken on a vortex mixer for 5 minutes and then heated at 95°C for 5 minutes to solubilize and denature tissue; this was repeated 3 times.
SDS polyacrylamide gel electrophoresis and Coomassie blue staining
Sample buffer (10 mM Tris, pH 6.8, 1 mM ethylenediaminetetraacetic acid (EDTA), 40 mM dithiothreitol (DTT), 0.005% Bromophenol Blue, 0.0025% Pyronin Yellow, 1% SDS, 10% sucrose) was added to all samples which were subsequently boiled for 10 minutes. 20 µg of sample from cases 1 and 2 of autopsy tissue and 20 µg of two representative PD DBS samples were resolved on SDS polyacrylamide gel electrophoresis (SDS-PAGE) using 15% polyacrylamide gels. Gels were stained with Coomassie blue R-250 to visualize protein and destained in 25% isopropanol/10% acetic acid as previously described 12.
Antibodies utilized for western blotting and immunofluorescence
For western blotting, anti-phosphorylated Ser129 (pSer129) αSyn rabbit monoclonal antibody EP1536Y was obtained from Abcam (Cambridge, MA). SNL-4 is a rabbit polyclonal antibody specific for αSyn residues 2–12 25. 3H11 is a mouse monoclonal antibody specific for the 43–63 residues of αSyn 2,26. 5C1 is a mouse monoclonal antibody specific for αsyn C-terminally truncated at residue 125 16. For immunofluorescence, neuronal specific rabbit anti-βIII tubulin antibody (T2200) was obtained from Sigma-Aldrich (St. Louis, MO).
Western blotting
Representative samples were chosen, and 5 µg of each sample (5 PD samples and 3 ET samples per blot) were loaded onto 15% polyacrylamide gels and resolved by SDS-PAGE, followed by electrophoretic transfer onto 0.2 µm pore nitrocellulose membranes (Bio-Rad, Hercules, CA) in carbonate transfer buffer (10 mM NaHCO3, 3 mM Na2CO3, pH 9.9) as previously described 12. Nonspecific binding of antibodies was blocked with 5% dry milk/Tris buffered saline (TBS) and membranes were incubated overnight at 4°C with primary antibody diluted in the same block solution. Membranes were washed in TBS and then incubated in goat anti-rabbit or goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (Jackson Immuno Research Labs, Westgrove, PA) and diluted in block solution for 1 hour at 25°C; immunocomplexes were detected with Western Lightning-Plus ECL reagents (PerkinElmer, Waltham, MA) followed by chemiluminescence detection (PXi, Syngene, Frederick, MD). Densitometry was performed via ImageJ software (NIH, Bethesda, MD) to quantify the signal for each sample. Western blot images are displayed with increased brightness and contrast for ease of viewing, with originals in supplementary information.
Purification of residual tissue for Hematoxylin & Eosin staining
For 5 DBS procedures on PD patients, tissue was purified from cannulas and microelectrodes in modified, non-denaturing conditions using PBS which allowed for hematoxylin & eosin (H&E) staining in 3 of the cases. Cannulas and microelectrodes were sectioned into ~ 1 cm fragments and placed into microcentrifuge tubes containing 150 µL of 1x PBS on ice. Samples were homogenized via manual pipetting, and then centrifuged at 1,500 x g for 5 minutes at 25°C. Cannula and microelectrode fragments were removed from the microcentrifuge tube and placed into a miniprep column and then centrifuged at 1,500 x g for 2 minutes at 25°C similar to the protocol detailed previously. For three of the samples, liquid sample was then transferred to a glass slide and 50 µL of 10% paraformaldehyde in 1x PBS was added for fixation overnight at 4°C. Samples were then rinsed in 1x PBS and placed into hematoxylin for staining, followed by aqueous washes. Samples were then stained in eosin followed by dehydration in ethanol, then xylene, then mounting and cover slipping. All slides were digitally scanned using an Aperio ScanScope CS instrument (40× magnification; Aperio Technologies Inc., Vista, CA), and images of representative areas of staining were captured using the ImageScope software (40× magnification; Aperio Technologies Inc.).
For the 2 PD samples not used for H&E, PBS purified tissue was homogenized and denatured through sequential boiling (95°C for 5 minutes) and shaking on a vortex mixer for 5 minutes repeated 3 times. This procedure was also utilized for 50 µL of tissue from case PD 20 for which the remainder of sample was used for H&E. Protein amount was then determined as described above using BCA assay, which was multiplied threefold for case PD 20 to account for the 100 µL used for H&E.
Immunofluorescence
For two DBS procedures on PD patients, tissue was purified from cannulas and microelectrodes and transferred to glass slides with paraformaldehyde fixation with the same protocol used for the H&E staining. Samples were then rinsed in 1x PBS and incubated with primary antibody overnight (4°C) diluted in 5% FBS/0.1M Tris (pH 7.6) followed with subsequent incubation for 1 hour at 25°C using secondary antibody (diluted in 5% FBS/0.1M Tris, pH 7.6) conjugated to Alexa-594 (Invitrogen). Samples were stained with 5 µg/mL 4′,6-diamindino-2-phenylindole (DAPI). Lastly, the sections were cover-slipped with Fluoromount-G (SouthernBiotech) and visualized using an Olympus BX51 microscope mounted with a DP71 Olympus digital camera to capture images at 40x magnification.
Quantitative analysis
All statistical tests and resulting graphs were conducted and created using GraphPad Prism (GraphPad software, La Jolla, CA). Comparison of protein amount between PD and ET samples purified using an SDS method was conducted using an unpaired T-test. Similarly, comparison of protein amount between all samples purified with SDS compared to the 3 using PBS was conducted using an unpaired T-test. For western blot data, bands were quantified using ImageJ software (NIH, Bethesda, MD) and densitometric comparisons between PD and ET bands for each antibody conducted using an unpaired T-test. For all figures, conventional terminology used for P-values is NS = no significance, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001.