Brain mediators of systemic oxidative stress on perceptual impairments in Parkinson’s disease

The study was approved by the Local Ethics Committee on Human Research of Kaohsiung Chang Gung Memorial Hospital in Taiwan. All participants or their guardians provided written informed consent prior to participation in the study. Twenty-seven right-handed PD patients (11 men and 16 women, mean age: 54.6 ± 9.3 years) with no previous history of neurological or psychiatric illnesses, psychotropic medication, or contraindication to magnetic resonance imaging (MRI) were prospectively enrolled at the Neurology Department of Kaohsiung Chang Gung Memorial Hospital. Patients were included if they had been diagnosed with idiopathic PD by an experienced neurologist according to the Parkinson’s Disease Society’s criteria for idiopathic Parkinson’s disease [19]. Of the 28 PD patients, ten had never used any anti-Parkinson’s medication, whereas the others used dopaminergic medication (levodopa and dopamine agonists). The disease severity and functional status of each patient were evaluated with the Unified Parkinson’s Disease Rating Scale (UPDRS) [20], the modified Hoehn and Yahr stages (HY-stage) [21], and the Schwab and England activities of daily living scale (SE-ADL) [22] in the “OFF” state, which refers to when levodopa appears to become less effective in eliminating motor symptoms and the patients are unable to function properly. The patients’ mean disease duration, defined as the time since the given patient subjectively noticed his or her first symptoms, was 4.1 ± 3.7 years. For comparison, 25 sex- and age-matched healthy subjects (11 men and 14 women, mean age: 50.9 ± 10.5 years) with no medical history of neurologic diseases or psychiatric illnesses, alcohol/substance abuse, or head injury, and with similar levels of education, were recruited.

We evaluated the oxidative stress condition in all subjects by measuring the serum concentration of TBARS and thiol. All subjects received blood sampling on the same day as the MRI study and neuro-psychological testing. Sera were isolated from peripheral blood samples drawn from each subject before and after the examination.

The serum TBARS were measured based on a well-established method for detecting lipid peroxidation [23]. The ability of anti-oxidative defense in response to increased oxidative damage was evaluated by measuring the serum level of total reduced thiols since serum thiols are physiologic free radical scavengers [24].

A clinical psychologist performed the neuro-psychological (NP) battery of tests from the Wechsler Adult Intelligence Scale-III (WAIS-III) [25]. The WAIS-III, with 14 subtests, allows us to summarize index scores and convert to different large ability areas reliably, including three IQs (Verbal IQ, Performance IQ, and Full Scale IQ) and four Indexes (Verbal Comprehension, Working Memory, Perceptual Organization, and Processing Speed).

In the current study, we focused on perceptual organization and processing speed functions in PD. These batteries comprise three tests for perceptual organization function and one test for processing speed function. The tests for perception organization function include picture completion, block design, and matrix reasoning tests. We sum the results from these tests to come up with an index score which represents the aggregate abilities of perceptual organization function.

The test for processing speed was a digit symbol-coding test which examines several cognitive processes of participants, such as detecting a test digit, searching for the corresponding symbol, and maintaining the symbol representation. We then further added scaled scores of perceptual organization and processing speed functions and converted them into the performance IQ.

Volumetric structural MRI scans were acquired on a GE Signa 3T whole-body MRI scanner (General Electric Healthcare, Milwaukee, WI, USA) using an eight-channel phase array head coil at the Kaohsiung Chang Gung Memorial Hospital in Taiwan. Whole brain three-dimensional T1 weighted images were collected for each participant using an inversion-recovery fluid-attenuated fast spoiled gradient-recalled echo pulse sequence with the following imaging parameters: repetition time (TR)/echo time (TE)/inversion time (TI) = 9.5/3.9/450 ms; flip angle = 15 degrees; number of excitations (NEX) = 1; field of view (FOV) = 240 × 240 mm²; matrix size = 512 × 512; voxel size = 0.47 × 0.47 × 1.3 mm³; and slice number = 110 axial slices (without interslice gaps). In order to identify any brain abnormalities, an additional volumetric axial T2-weighted fast spin-echo sequence (TR/TE = 4200/102 ms; echo train length = 18; NEX = 2; FOV = 240 mm²; slice thickness = 5 mm; matrix size = 320 × 256 and 18 slices) and axial T2-weighted inversion-recovery fluid-attenuated sequence (TR/TE/TI = 8000/100/2000 ms; NEX = 1; FOV = 240 mm²; slice thickness = 5 mm; matrix size = 320*256 and 18 slices) were used in the same imaging session.

T1-weighted structural MRI scans were analyzed with voxel-based morphometry (VBM) [26] using the VBM8 toolbox (http://​dbm.​neuro.​uni-jena.​de), which was implemented using the Statistical Parametric Mapping software program (SPM8, Wellcome Institute of Neurology, University College London, UK, http://​www.​fil.​ion.​ucl.​ac.​uk/​spm/​) with default settings. All the image processing procedures, including tissue segmentation, study-specific template construction, and spatial normalization, were conducted according to a previous study [27] and are summarized in the Additional file 1 for the present study.

The demographic and clinical data of the participants are shown in Table 1. In the ANCOVA analyses, the patients with PD had worse performance on the perceptual function tests, including the picture completion (P = 0.007), block design (P = 0.002), matrix reasoning (P = 0.020), and digit symbol-coding tests (P < 0.001). Further analysis showed that the patients with PD had a worse mean score in terms of the perceptual organization index (P = 0.004). Moreover, when that mean was combined with the mean score for digital symbol-coding, the mean performance IQ in patients with PD was also found to be worse than the mean for the healthy controls (P = 0.002).

The mean serum concentration of TBARS was significantly higher in the PD patients than in the controls (P < 0.001). The mean serum concentration of free thiol was lower in PD patients than in the controls, but the difference failed to achieve statistical significance (P = 0.191). Since only the serum TBARS level revealed a significant group difference, the following correlation and mediation analyses were primarily focused on and carried out with regard to the TBARS levels.

Compared to healthy controls, PD patients showed a significant GM volume reduction in the following brain regions: (1) bilateral temporal gyri (middle temporal gyrus and fusiform gyrus), (2) bilateral frontal gyri, (3) limbic lobe (hippocampus and uncus), (4) left inferior parietal lobule, (5) right caudate nucleus, and (6) insula. We did not find increased GM volume in PD patients compared with the healthy controls (Additional file 2: Table S2; Fig. 2).

Consistent with our hypotheses, elevated serum oxidative stress levels were associated with poorer performances on several perceptual subtests, which suggested that systemic inflammation might degrade general perceptual functions directly or indirectly in PD (Table 2). We found that higher TBARS levels were associated with worse matrix reasoning (p = 0.017, r = −0.337), digit symbol (p = 0.044, r = −0.286), and perceptual organization index (p = 0.035, r = −0.299) scores. We also found that high TBARS levels were borderline significantly correlated with worse block design (p = 0.066, r = −0.262), picture completion (p = 0.058, r = −0.270), and performance IQ (p = 0.062, r = −0.266) scores.

Consistent with our hypothesis and in line with the extant literature, patients with PD experienced higher serum oxidative stress and worse perceptual functions. Furthermore, we identified gray matter atrophy throughout much of the cortex, including anatomical locations that typically appear to be particularly sensitive to the effects of inflammation, such as the temporal lobe, hippocampus, and insula [30]. We further demonstrated, for the first time, that gray matter volume atrophy in the temporal lobe, hippocampus, insula, and parietal lobe mediate the relationships between TBARS levels and general perceptual function scores. This suggests that higher oxidative stress levels are associated with poor perceptual functions by means of smaller temporal and mesolimbic volumes and that those regions support the retention of visual memories, the processing of sensory inputs, the comprehension of language, the storing of new memories and emotions in a manner that may differ from the way in which the prefrontal cortex supports executive functioning in PD.

Studies have demonstrated that an imbalance in pro-oxidant/antioxidant homeostasis can enhance the generation of toxic reactive oxygen species [31] and may be related to disease severity in PD [32]. The TBARS level is one of the markers of oxidative stress, and has been found to be elevated not only in the substantia nigra but also in the CSF and plasma in PD [33‐35]. In addition, many cytokines, as well as TBARS, are also markers of reactive oxygen species (ROS) activity, which can worsen central neuron cell injury through the elevation of reactivated microglia in the substantia nigra [36]. Cytokines produced in the brain can freely diffuse past the blood–brain barrier (BBB), a phenomenon which has been demonstrated to induce memory impairments [37]. Our study supports the previous hypothesis that oxidative stress is implicated in the pathogenesis of PD.

In addition to the nigrostriatal region, inflammation activated microglial cells in PD were also observed in various brain regions, such as the hippocampus, temporal lobe, and other regions of the cerebral cortex [30]. Actually, increased oxidative stress/inflammation has also been found to be associated with temporal lobe damage in aging [38] due to atherosclerosis and Alzheimer’s disease [39], with the pathological accumulation of amyloid and neurofibrillary tangles. Two leading factors might contribute this temporal lobe vulnerability: relatively high receptor and messenger RNA expression for proinflammatory cytokines [40] in those regions and higher links to portions of the salience network, such as the insula, which has a role in regulating the immune system and can enhance the peripheral inflammatory conditions [41] and subsequently affect the neural organization of semantic memory [42]. From animal studies, it has been demonstrated that injections of inflammatory cytokines into the hippocampus and the overexpression of IL-1 messenger RNA in the hippocampus selectively deteriorate spatial and contextual memory processes [43]. In addition, elevated peripheral inflammation can stimulate IL-1 expression within the central nervous system, including the medial temporal lobe, and generates increased cytokine expression with subsequent impacts on temporal lobe-dependent memory [44]. The vagus nerve, which serves as a neurally mediated immune-brain pathway, can also enhance hippocampal activity by peripheral inflammatory challenge and electrical stimulation [45, 46]. Taken together, these data demonstrate the sensitivity of human medial temporal lobe structures to systemic oxidative stress/inflammation and provide mechanistic insights relevant to the broader literature linking severe or chronic inflammation to the attrition of human memory.

In a previous cortical thickness analysis of PD with mild cognitive impairment, patient groups revealed widespread cortical thinning compared with controls, including thinning of the right inferior temporal gyrus, left superior parietal cortex, precuneus, lateral occipital, temporal gyri anterior cingulate, and superior frontal gyri [1]. The same study also found that memory and visuospatial performance, the core feature of perceptual function, were associated with temporoparietal and superior frontal gyri thinning. Our results replicated these previous findings and also consistently demonstrated the interaction between perceptual functional impairment and volume atrophy in the middle temporal gyrus, inferior parietal lobe, hippocampus, and insula. Although fewer cortical regions were found to be involved in this study, the relatively small sample size and younger patient group might have led to these slightly different results. Furthermore, different gray matter quantitative morphometrical evaluation methods which are sensitive to different properties of gray matter tissue (i.e., volume vs. cortical thickness) might also have led to the different outcomes. We do believe, however, that the results of the current study, together with those of the previous investigation, indicate that particular cortical atrophy can serve as a marker for perceptual function decline in early PD.

The test of mediation amounts to a test of whether controlling for each brain mediator explains a significant amount of the covariance between the independent variable and the dependent variable. In the initial analyses, we found that higher serum TBARS levels were associated with worse perceptual functions. We further examined whether the relationship between serum TBARS levels and perceptual functions was explained indirectly by greater damage to perceptual functional specific anatomies in patients with PD using mediation analyses. We found that the middle temporal lobe and insula, as well as the hippocampus, play the central roles in mediating most of the TBARS-perceptual function relationships. Interestingly, the caudate nucleus and frontal lobe volume reduction, which is a hallmark of PD, failed to mediate the association between oxidative stress and perceptual function. Actually, perceptual impairment in PD occurs not only on visuomotor tasks requiring a complex motor response such as drawing but also on visuoperceptual tasks that require a limited motor response. Furthermore, this perceptual impairment is not related to a decrement in intellectual abilities and can be clearly distinguished from dementia [4]. In the present study, though the results could not exclude the influence of TBARS on other aspects of systemic status, they suggest that TBARS may play a particular role in the perceptual deficits observed in PD. Another explanation might be the ceiling effect, which suggests that inflammation occurs earlier in the striatum and then is followed by inflammation in the temporal lobe and hippocampus, resulting in the sequential clinical presentations progressing from movement disorders to cognitive impairments. These results, however, should be interpreted with caution.

The interpretation of the findings here must be tempered by some of the limitations of the present study. We do not know the temporal relationships between peripheral inflammation, symptomatology, and anatomical integrity. Further task design or longitudinal evaluation with manually controlled systemic oxidative stress levels in animal models should be carried out in the future. In addition, the measurement of only a few biomarkers of oxidative damage cannot be considered a valid tool for exploring the multifaceted, complex oxidant/antioxidant imbalance in PD. Furthermore, the oxidant/antioxidant balance of PD patients may be influenced by a multitude of parameters. The interactions between the CNS and systemic oxidative stress and the effect of individual genetic variations and physical exercise could also affect either the systemic oxidative stress or the brain volume, and these interactions were not well evaluated in the present study. While systemic oxidative stress is also likely to have widespread effects on the brain tissue, the specific anatomical volumes that mediate a given cognitive function are likely to differ depending on the examinations being assessed [47]. In addition, recent research suggests that network-based rather than regional anatomic involvements contribute to the neurodegeneration and behavioral differences seen in PD, such that assessing structural or functional connectivity between brain regions associated with these phenotypes would clarify the complex neural network involved in oxidative stress–cognition relationships.