Neurochemical Characterization of A53T-Alpha-Synuclein and 6-Hydroxydopamine Rat Models for Parkinson’s Disease through Animal PET Imaging Analysis

Abstract

Objective

In preclinical research of Parkinson’s disease, several rodent models, notably the classical 6-hydroxydopamine (6-OHDA) model and the A53T-alpha-synuclein model, have been widely used, yet their distinct neurochemical characteristics in conjunction with behavioral and histopathological changes have been scarcely documented.

Methods

We examined the two rat models of Parkinson’s disease and characterized them using [¹⁸F]fluoropropyl-carbomethoxyiodophenyltropane (FP-CIT) animal positron emission tomography (PET) imaging. The 6-OHDA model (n=10) was induced by unilateral injection of 6-OHDA into the middle forebrain bundle, while the A53T-alpha-synuclein model (n=10) was mediated by the adeno-associated viral vectors injected into the substantia nigra. We hypothesized that these models would present differential neurochemical profiles, which could reflect their behavioral and histopathological features and potentially serve as a supplementary tool for evaluating the outcomes of interventions in animal experiments.

Results

The striatum showed decreased PET uptake on the affected side compared to the unaffected control side, which was highly correlated with the stepping behaviors (R=0.854; 95% confidence interval [CI], 0.606 to 0.951). The decrease in striatal PET uptake was more pronounced in the 6-OHDA model than in the A53T-alpha-synuclein model : the 6-OHDA model exhibited a 60% decrease (95% CI, 48% to 65%) in the affected side compared the control side, while the A53T-alpha-synuclein model exhibited a 20% decrease (95% CI, -16% to 47%). Interestingly, PET uptake in the forebrain cortical region, including the motor cortex, was exclusively decreased in the 6-OHDA model (p=1.0×10^-4 and p=1.2×10^-3, respectively), indicating that 6-OHDA model is affected not only in the nigrostriatal system but also in other cortical regions. Conversely, the A53T-alpha-synuclein model showed no significant alterations in these cortical regions.

Conclusion

Although the A53T-alpha-synuclein model demonstrates less definitive behavioral changes compared to the 6-OHDA model, it presents a more confined pathophysiological representation of Parkinson’s disease and may be better suited for evaluating certain therapeutic interventions when utilized with adequate neurochemical characterization.

INTRODUCTION

Parkinson’s disease is the most prevalent alpha-synucleinopathy leading to a progressive neurodegenerative disease, which primarily manifests as involuntary movement disorders but progresses to include additional neurocognitive impairments in advanced stages. In preclinical research, various animal models have been developed to investigate the pathophysiology of Parkinson’s disease. The classical neurotoxin-induced rodent model, represented by 6-hydroxydopamine (6-OHDA), reproduces the rapid chemical destruction of catecholaminergic neurons and has been extensively utilized in numerous studies over the past half-century [8,10,19]. More recent research has introduced alpha-synuclein overexpression models to mimic the pathological processes of Parkinson’s disease [12,13,16,18] These can be mediated by viral vector plasmids via stereotactic intracerebral injections to reproduce local somatic expression in targeted brain structures [16]. The delivery of A53T-mutant human SNCA leads to the accumulation of alpha-synuclein and subsequent nigrostriatal dopaminergic dysfunction [12]. These animal models, neurotoxin-induced or viral vector-mediated, are essential for understanding the impacts of therapeutic intervention on Parkinson’s disease, serving respectively as complete or partial models of dopaminergic deficits [13,14].

Dopamine active transporter (DAT) imaging, as represented by N-(3-[¹⁸F]fluoropropyl)-2β-carbomethoxy-3β-(4-iodophenyl) nortropane ([¹⁸F]FP-CIT) positron emission tomography (PET) scan, is a molecular imaging technique using a radioactive tracer that is particularly specific to dopaminergic neurons. With the advancement of dedicated small-animal PET systems, the application of DAT imaging in rodent Parkinson’s disease models as a supplementary research tool has increased over the past decade [11]. Despite this, comprehensive investigations that correlate these imaging studies with assessments of animal behavioral changes remain sparse. Since rat and mouse brains are 600- and 2400-fold smaller than a human brain, respectively, the spatial resolution of preclinical scanners could be limited and poses substantial challenges to the utilization of molecular imaging in rodent models [9].

However, the integration of small-animal PET with high-resolution magnetic resonance imaging (MRI) systems enables the simultaneous acquisition of structural data from MRI and neurochemical information from PET with enhanced spatial resolution, potentially enabling the quantitative analysis of PET scan images on an anatomical basis. We hypothesized that this quantitative analysis could effectively reflect the severity of behavioral changes and histopathological alterations in rat models, owing to their relatively large tissue size compared to mouse models. Therefore, we investigated the characteristics of neurochemical imaging in two distinct rat models of Parkinson’s disease, using the [¹⁸F]FP-CIT PET. The goal of this study is to explore the feasibility of using this approach to evaluate the outcomes of interventions in animal experiments.

MATERIALS AND METHODS

Study approval

This study received approval from the Ethics Committee for Animal Experiments (project number, 2023-10-223), and all animal procedures complied with the guidelines of the Institutional Animal Care and Use Committee. All subjects included in this research were initially designated for the control groups of other projects, thus no animals were specifically sacrificed for the purposes of this study.

Study subjects

A total of 20 male Wistar rats, weighting 300–350 g at the start of the experiment, were enrolled in this study. The subjects were divided into two experimental groups : half received a unilateral injection of pAAV-DJ-hSNCA (A53T), an adeno-associated viral vector serotype DJ containing A53T-mutant human SNCA, into the substantia nigra for overexpression of alpha-synuclein (‘A53T-α-Syn’ model, n=10), while the other half received a unilateral injection of 6-OHDA into the medial forebrain bundle to achieve complete nigrostriatal dopaminergic denervation (‘6-OHDA’ model, n=10). The lesioning procedure was performed on the right side, with the left side serving as a control.

Behavioral assessment

The subjects were housed in a 12-hour light/dark cycle with unrestricted access to food and water. For behavioral assessment, the treadmill stepping test and cylinder test were conducted weekly to evaluate the severity of forelimb akinesia, including assessments 1 week preoperatively and 3 weeks postoperatively.

During the stepping test, the experimenter held both hindlimbs and one forelimb of the subject, while allowing the other forelimb to spontaneously touch the treadmill track (Fig. 1A). The affected and control sides of forelimbs were alternately tested for 10 seconds each at a speed of 0.2 m/s, with each test repeated twice per session. Results were quantified by counting the number of adjusting steps for each side from video recordings, and then averaged across the two trials.

Fig. 1.

The treadmill stepping and cylinder tests for behavioral assessment in rodent Parkinson’s disease models. A : Illustration of the treadmill stepping test. B : The relative stepping ratios of the affected forelimb compared to the sum of those trials from each forelimb, calculated as [count of the affected side] / ([count of the affected side] + [count of the control side]). C : Illustration of the cylinder test. D : The relative counts of wall contacts compared to the sum of those trials from each forelimb. **p<2.5×10^-3, ***p<2.5×10^-4. 6-OHDA : 6-hydroxydopamine.

For the cylinder test, each subject was placed in a transparent cylindrical container with a diameter of 20 cm and height of 35 cm to assess spontaneous forelimb use during vertical exploratory movements (Fig. 1C). As the rat moved upwards along the cylinder wall, the use of each forelimb was recorded during the first 20 wall contacts. Results were presented as the ratio of the affected forelimb usage to the total count, representing the limb-use asymmetry.

Stereotactic procedures for model establishment

Surgical procedures for the delivery of viral vector plasmids or neurotoxins were performed under general anesthesia, administrated via an intraperitoneal injection of a 1 : 1 mixture of zolazepam/tiletamine at 35 mg/kg, combined with xylazine at 5 mg/kg. Subjects were then positioned on a rodent stereotaxic instrument (Harvard Apparatus, Holliston, MA, USA).

For A53T-α-Syn model, 2.5 μL of pAAV-DJ-hSNCA (A53T) vector plasmid (KIST, Seoul, Korea), prepared at a concentration of 2×10¹³ genome copies/mL, was injected into the right substantia nigra. The target coordinate was set to 5.4 mm posterior to bregma, 2.0 mm lateral to the midline, and 7.5 mm ventral to the dura, based on the Paxinos-Watson rat brain atlas. For 6-OHDA model, 8 µg of 6-OHDA (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 4 µL of saline with 0.1% ascorbic acid, was injected into the right medial forebrain bundle at target coordinate of 2.2 mm posterior to bregma, 1.5 mm lateral to the midline, and 8.0 mm ventral to the dura, with the incisor bar set to 4.5 mm above the interaural line [15]. The intracerebral injections were administered at a rate of 0.2–1.0 μL/min using a 33-gauge Hamilton syringe operated by an automated micro syringe pump (Harvard Apparatus), with the syringe needle left in place for 5 minutes after the injection to prevent backflow, and then slowly retracted over the next 5 minutes.

For the verification of complete lesioning of the 6-OHDA models [3], we performed the apomorphine-induced rotation test at 2 weeks after 6-OHDA injection. In this test, each subject was tethered to a rotameter system (Panlab, Barcelona, Spain), shortly after subcutaneous injection of apomorphine at 0.25 mg/kg. Both ipsiversive and contraversive rotations were counted for 45 minutes, and the result was presented as average net rotations per minute, calculated as subtracting ipsiversive rotations from contraversive rotations. Subjects exhibiting more than six contraversive rotations per minute were considered complete Parkinson’s disease models.

PET-MR image acquisition and quantitative analysis

For each subject, T1- and T2-weighted structural MRI and [¹⁸F]FP-CIT PET scans were performed using an animal PET/MRI system (nanoScan; Mediso Medical Imaging Systems, Budapest, Hungary) 4 weeks after the stereotactic lesioning procedure. The subjects were under general anesthesia using isoflurane and were injected with 15 MBq [¹⁸F]FP-CIT radiotracer in 0.2 mL of sterile saline intravenously 30 minutes prior to scanning and scanned for static 20 minutes.

Structural brain MRI scans were acquired in the coronal plane with a slice thickness of 0.7 mm. The field of view was set at 60 mm, achieving an in-plane resolution of 0.27×0.27 mm per pixel. The scanning parameters included a T1-weighted gradient echo sequence with a repetition time (TR) of 15 ms and echo time (TE) of 3 ms, and a T2-weighted fast spin echo sequence with a TR of 2400 ms and a TE of 110 ms. The PET scans were acquired for three-dimensional full-detect mode with an energy level of 250–750 keV.

The scanned PET data were reconstructed using the imaging platform provided by the manufacturer and postprocessed for normalization to generate standardized uptake values (SUVs) based on each subject’s weight and the administered dose of the radiopharmaceutical. The total time from radiopharmaceutical injection to the start of the scanning session, along with the duration of the frame scanning, was used to calculate the decay correction factor based on the known half-life of [¹⁸F] (109.8 minutes). The dose was adjusted for radioactive decay using this decay factor and the positron fraction of the radionuclide (96.7%).

PET and MR images of each subject were resampled to a resolution of 0.1 mm isovoxel spacing with b-spline interpolation and aligned to the coordinate system of the Paxinos-Watson atlas. Segmentation labels for each subject were derived from the existing in vivo T2-weighted anatomical template and atlases for the Wistar rat brain [1]. This segmentation process utilized an affine transformation followed by SyN registration from the template to each subject, executed using ANTs in Python. The specific binding ratios (SBRs) for each region of interest from the segmentation atlas were calculated using the cerebellum as a reference, and results were presented as the median value for each region of interest in each subject.

Tissue processing and immunohistochemical staining

Experimental animals were subjected to transcardiac perfusion with saline containing 10 units/mL heparin, followed by fixation with a 4% paraformaldehyde solution in phosphate-buffered saline (PBS). Brain tissues were then extracted and preserved in the fixation solution for 24 hours, followed by cryoprotection through dehydration in 30% sucrose until fully submerged. The 40 µm-thick coronal sections of the substantia nigra (anterior-posterior [AP] -4.8 mm to -6.0 mm from bregma) and the striatum (AP +2.0 mm to -0.1 mm) were collected using a cryostat (Leica, Wetzlar, Germany) and stored in PBS containing 0.08% sodium azide at 4°C.

Immunofluorescence staining was performed on the serial coronal sections using tyrosine hydroxylase (TH) as a marker for dopaminergic neurons. The sections were washed in PBS containing 0.5% bovine serum albumin (BSA) and then incubated in blocking solution containing 0.5% BSA, 0.2% Triton X-100, and 0.08% sodium azide in PBS. They were subsequently incubated overnight with primary antibodies (anti-TH mouse monoclonal antibody [mAb], 1 : 2000, Sigma-Aldrich; or anti-TH rabbit mAb, 1 : 1000; Abcam, Cambridge, UK) in PBS containing 0.5% BSA. This was followed by a two-hour incubation with the secondary antibody (AlexaFluor-555 donkey anti-mouse IgG, 1 : 1,000; Invitrogen, Carlsbad, CA, USA). The fluorescent-labeled tissues were coverslipped with a fluorescent mounting medium (DAKO, Glostrup, Denmark). Fluorescent images were obtained using a confocal microscope (Carl Zeiss, Oberkochen, Germany). For nigral and striatal cell counting, TH-immunopositive cells were reviewed in three subsequent coronal sections. Results were presented as the percentage of TH-positive cells in the lesioned tissue compared with the number of TH-positive cells on the control side.

Statistical consideration

The resulting data were presented as median with interquartile ranges unless stated otherwise. Differences between the experimental groups were assessed using an unpaired t-test. Statistical significance was defined as a p-value of less than 0.05, followed by a Bonferroni correction. Associations between neurochemical imaging features and behavioral outcomes were analyzed using Pearson’s correlation coefficients (R). The significance of these correlations was assessed by calculating 95% confidence intervals (CIs) using the Fisher transformation, whereby correlations were considered significant if the CIs did not include zero. The percentage decrease in SUVs or SBRs of a region of interest on the affected side compared to the contralateral, unaffected control side was determined by computing the ratio of the medians between two subgroups. The results were reported with their corresponding 95% CIs, which were estimated using the bootstrapping method. All computational analyses were conducted using Python and R, employing relevant packages.

RESULTS

Model establishment and behavioral outcomes

All 20 subjects were successfully established in either A53T-α-Syn and 6-OHDA models. The subjects’ median weight was 376 g (343–410) at the time of the operation and 441 g (420–467) at the time of the imaging study. In the initial stepping test prior to the procedure, subjects in both groups exhibited a median of 33.5 (31.9–34.6) steps per 10 seconds as baseline. After the model establishment, subjects in the A53T-α-Syn model exhibited a median of 8.3 (5.5–9.9) steps on the affected side, compared to 28.8 (27.0–31.4) steps on the unaffected control side. In contrast, subjects in the 6-OHDA model displayed 1.8 (1.0–3.0) steps on the affected side and 33.8 (33.0–34.9) steps on the control side, presenting more severe forelimb akinesia (Fig. 1B). In the cylinder test, the A53T-α-Syn model subjects showed 8.0 (7.0–8.5) wall contacts for the affected side and 12.0 (11.5–12.5) for the control side. On the other hand, the 6-OHDA model subjects showed 5.0 (3.5–5.8) wall contacts for the affected side and 15.0 (14.2–16.5) for the control side, presenting predominant asymmetry in the forelimb use (Fig. 1D). The preoperative initial baseline of both models was 10.0 (10.0–11.0) and 10.0 (9.0–11.0) wall contacts, respectively. In summary, the motor function in affected side were significantly more severely decreased in 6-OHDA model compared to A53T-α-Syn model.

Neurochemical characteristics of the rat Parkinson’s disease models

PET/MRI imaging studies were successfully conducted for 17 subjects (n=7 in the A53T-α-Syn model and n=10 in the 6-OHDA model), except three subjects whose conditions were not tolerable to the general anesthesia. The total dose of radionuclide was 32.7 MBq/kg (31.6–34.9) and the time from radiopharmaceutical injection to the end of PET acquisition was 51.0 minutes (49.0–53.0). The median SUV of the cerebellum was measured at 0.79 (0.74–0.82).

Striatal PET uptakes

DAT imaging demonstrated distinct striatal PET uptake across the 17 subjects. The median SUVs of the striatum were 1.11 (0.93–1.67) on the affected side, compared to 2.37 (2.16–2.59) on the control side. The striatal SUV ratios of the affected side compared to the control side in each subject were positively correlated with the severity of the subject’s akinetic behavior, as evaluated by the relative stepping ratios (R=0.854; 95% CI, 0.606 to 0.951). The distinction in the striatal SUVs on the affected side was clear between the A53T-α-Syn and the 6-OHDA models : 1.92 (1.63–2.32) in the A53T-α-Syn model and 0.93 (0.86–1.11 in the 6-OHDA model; p=2.4×10^-4). The decreases in median striatal SUVs compared to their respective control side were 20% (95% CI, -16% to 47%) in the A53T-α-Syn model and 60% (95% CI, 48% to 65%) in the 6-OHDA model. The A53T-α-Syn model showed a partial reduction in PET uptake within the affected striatum, indicating a less extensive dopaminergic loss. Conversely, the 6-OHDA model showed a complete loss of striatal PET uptake on the affected side, suggesting a more severe degeneration of dopaminergic neurons. The substantia nigra did not show a significant difference between the two models, nor on the control side, attributed to the challenges in detecting signal differences within the small midbrain region of the rats : 1.09 (1.03–1.24) on the affected side of the A53T-α-Syn model, 1.12 (1.03–1.15) on the affected side of the 6-OHDA model, and 1.08 (1.03–1.28) on the control side.

Cortical PET uptakes

The neurochemical profiling from PET uptake across various brain regions beyond the striatum provided additionl findings (Fig. 2). Interestingly, the 6-OHDA model displayed a significant decrease in PET uptake on the affected side in the various cortical regions, such as the motor cortex, somatosensory cortex, insular cortex and cingulate cortex, highlighting the extensive impact of the neurotoxin-based model. These differences in the motor and cingulate cortices were particularly noticeable, as these structures are less proximate to the striatum compared to the somatosensory and insular cortices. The median SBRs of the motor cortex on the affected side were 1.07 (1.03–1.14) in the 6-OHDA model compared to 1.35 (1.31–1.42) in the A53T-α-Syn model (p=1.0×10^-4), exhibiting a 20% decrease (95% CI, 12% to 27%). Similarily, the SBRs of the cingulate cortex were 1.10 (1.07–1.19) in the 6-OHDA model, compared to 1.36 (1.29–1.39) in the A53T-α-Syn model (p=1.2×10^-3), exhibiting a 19% decrease (95% CI, 7% to 24%). In A53T-α-Syn model, the PET uptakes in all thse regions were preserved and comparable to the control side, except the striatum.

Fig. 2.

Neurochemical profiling and comparative analysis in distinct regions of interest within the two rat models of Parkinson’s disease. A : The standardized uptake values from positron emission tomography (PET) images across various anatomical regions of interest are shown with separate presentations for the affected (right) and control (left) sides, revealing distinct PET uptake profiles. B : Comparative analysis of PET imaging features in each regions or interest demonstrates that the 6-hydroxydopamine (6-OHDA) model presents a significantly greater decrease in PET uptake levels in the multiple forebrain regions beyond the striatum, including the cingulate, motor, somatosensory, and insular cortices. Values represent specific binding ratios referenced tof median uptake of the cerebellum, plotted on a logarithmic scale. *p<0.013, **p<2.5×10^-3, ***p<2.5×10^-4.

Immunohistochemical findings

In immunofluorescent imaging, TH-positive cells at the striatum were relatively abundant in the A53T-α-Syn model, whereas the 6-OHDA model showed extensively decreased TH staining in the striatum, consistent with the striatal uptake observed in the PET scans (Fig. 3). Interestingly, a considerable loss of TH-positive neurons was observed at the substantia nigra level in the A53T-α-Syn model compared to the control side, 68.1% (15.1–88.7%), suggesting that A53T-α-Syn overexpression causes inhibition in the substantia nigra region and a reduction of TH expression but does not thoroughly affect axon terminals in the striatal level. Sections of the substantia nigra in the 6-OHDA model showed a nearly complete loss of TH-positive neurons, 88.2% (78.0–94.7%), indicating that the 6-OHDA model presents complete lesions, while the A53T-α-Syn model presents partial lesions.

Fig. 3.

Dopamine active transporter (DAT) positron emission tomography (PET) scan and immunofluorescence confocal images of rat Parkinson’s disease models. Coronal sections of [¹⁸F]fluoropropyl-carbomethoxyiodophenyltropane (FP-CIT) PET images and confocal images (100× magnification) of tyrosine hydroxylase (TH) immunofluorescence in the striatum (A and C; anterior-posterior [AP] +1.8 mm from bregma) and in the substantia nigra (B and D; AP -5.2 mm) are shown. The region of interest was delineated by the white boxes in PET images. In the immunofluorescence, the numbers of TH positive cells in substantia nigra (D) and the intensity of TH positive axon terminal in the striatum (C) were both significantly decreased in the 6-hydroxydopamine (6-OHDA) model compared to A53T-α-Syn, representing the complete nigrostriatal dopaminergic degeneration. In DAT imaging, the A53T-α-Syn model exhibited a partial loss of PET uptake in the affected striatum, distinct with the complete loss observed in the 6-OHDA model (A). The confocal images were quantitatively analyzed, where data were presented as a relative cell survival ratio of the affected side compared to those measured in the both sides (E). On the other hand, PET uptake in the substantia nigra (B) was not feasible to demarcate a discernible change within the midbrain region due to the limited spatial resolution. **p<0.01, ***p<0.001.

DISCUSSION

In this study, we observed that PET uptake in rat Parkinson’s disease models positively correlates with both behavioral and histopathological results. Specifically, the severity of the decrease in striatal PET uptake reflects the corresponding stepping behaviors of the affected forelimbs in rats, as indicated by a strong correlation with a correlation coefficient of 0.854 (95% CI, 0.606 to 0.951). This finding aligns with existing clinical research indicating that quantitative analysis of DAT imaging has been applied not only for the diagnosis of Parkinson’s disease but also as a reliable indicator of the disease’s severity [4,5,7,21,22,25,26]. In addition, the striatal PET uptake values were significantly different in the two representative rodent Parkinson’s disease models, corresponding to the findings from the TH-immunofluorescent imaging that showed complete nigrostriatal dopaminergic denervation in the 6-OHDA model compared to partial loss in the A53T-α-Syn model. These results were consistent with the striatal SUVs, showing a 60% decrease (95% CI, 48% to 65%) in the 6-OHDA compared to a 20% decrease (95% CI, -16% to 47%) in A53T-α-Syn model.

Additionally, we demonstrated the distinct neurochemical profiles of these two rat models beyond the nigrostriatal system. PET uptakes in various cortical regions of the forebrain was noticeably decreased in the 6-OHDA model, suggesting that 6-OHDA induces more extensive destruction throughout various cerebral regions. These findings align with recent insights indicating that 6-OHDA affects not only dopaminergic neurons but also involves other catecholaminergic systems [24]. In the 6-OHDA model, that non-dopaminergic structures can be affected by extracellular processes, presumably resulting from the formation of reactive oxygen species. Furthermore, the value of 6-OHDA model is constrained by its basis on an acute toxin, while Parkinson’s disease develops relatively slowly. This suggests that the A53T-α-Syn model can offer a more confined pathophysiology representative of Parkinson’s disease in terms of neurochemical changes compared to the 6-OHDA model, making it better suited for evaluating therapeutic interventions for Parkinson’s disease.

For neurochemical characterization of Parkinson’s disease model, various radiotracers have been introduced in the existing literature [20,23]. In particular, brain metabolism has been frequently evaluated using 2-[¹⁸F]-fluoro-2-deoxy-D-glucose ([¹⁸F] FDG), which has consistently shown hypometabolism in both striatal and cortical regions on the lesioned side of the 6-OHDA model, correlating with forelimb gait patterns [17]. For the evaluation of dopamine depletion, 6-[¹⁸F]f luoro-3,4-dihydroxy-L-phenylalanine ([¹⁸F]FDOPA) and 6-[¹⁸F]fluoro-L-m-tyrosine ([¹⁸F]FMT) have been utilized, with the latter reported to be better in characterizing partial nigrostriatal denervation [2]. However, there has been a relatively lack of documentation on the neurochemical characteristics directly comparing the classical 6-OHDA model and the A53T-α-Syn model.

In our findings, [¹⁸F]FP-CIT PET also exhibited differential cortical uptake patterns beyond nigrostriatal system between the two models. Decreased PET uptake was observed not only in the striatum but also in the motor and cingulate cortices in the 6-OHDA model, whereas the A53T-α-Syn model exhibited a confined decline within the striatal uptake. This difference might result from the varied perfusion of respective cortical regions indicated by [¹⁸F]-radiotracer, possibly caused by the extensive chemical destruction induced by 6-OHDA. An additional role of [¹⁸F]FP-CIT is its potential to represent early cerebral perfusion due to the high membrane permeability of the radiotracer, as well as its high specificity for the desired dopaminergic structures [9]. Therefore, it can be utilize to characterize differential cortical activities, similar to [¹⁸F]FDG, which has been employed in some clinical studies to explain the diverse manifestations of Parkinson’s disease [16]. We also observed decreased PET uptake in the insular and somatosensory cortices (Fig. 2B); however, we consider this phenomenon might be a false positive due to their close anatomical proximity to the striatum.

One limitation of this study is the insufficient characterization of certain anatomical regions using the current animal PET imaging system, likely due to its limited spatial resolution (Fig. 3B). Although the striatal and cortical PET uptakes fairly reflected the behavioral outcome, this methodology did not seem feasible enough to address molecular changes within the substantia nigra. Additionally, our experiments conducted PET scans at relatively early stages, four weeks after the stereotactic injection, which may not be sufficient to observe temporal changes in the dopaminergic system beyond the striatum in the A53T-α-Syn model [6]. Given the current knowledge of progressive alpha-synuclein accumulation in advanced Parkinson’s disease, it might be necessary to analyze neurochemical characteristics thought the late stage of the model to better represent the chronic disease progression. Nevertheless, our approach provided meaningful findings through PET imaging features, explaining more severe forelimb akinesia in the classical 6-OHDA model compared to the A53T-α-Syn model in the early stage. These non-invasive molecular imaging techniques might be useful in preclinical research for validating animal disease models and assessing the potential efficacy of interventional drugs.

CONCLUSION

Our findings, supported by distinct neurochemical characteristics, indicate that the A53T-alpha-synuclein model presents a more confined pathophysiological representation of Parkinson’s disease compared to the 6-OHDA model, despite representation fewer behavioral and histopathological changes. This suggests that the A53T-alpha-synuclein model may be better suited for evaluating certain therapeutic interventions.