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Tear levels of tumor necrosis factor-alpha in patients with Parkinson's disease
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease
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Selim Selc¸uk C¸omo˘glu a , Hayat Güven a,∗ , Mutlu Acar b , Gülfer Öztürk c , Bilge Koc¸er a
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Ankara Dıs¸kapı Yıldırım Beyazıt Training and Research Hospital, Department of Neurology, Turkey Ankara Dıs¸kapı Yıldırım Beyazıt Training and Research Hospital, Department of Ophthalmology, Turkey Ankara Dıs¸kapı Yıldırım Beyazıt Training and Research Hospital, Department of Biochemistry, Turkey
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h i g h l i g h t s • TNF-␣ and other pro-inflammatory cytokines can induce dopaminergic neuron loss. • We aimed to determine TNF-␣ levels in the tears of patients with PD. • Tear TNF-␣ values were significantly higher in patients with PD than in control subjects.
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Article history: Received 12 June 2013 Received in revised form 7 August 2013 Accepted 12 August 2013
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Keywords: Tumor necrosis factor-alpha Neuroinflammation Parkinson’s disease Tear
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1. Introduction
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Neuroinflammatory events mediated by the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-␣) cause progressive neurodegeneration in dopaminergic neurons, and play an important role in the pathogenesis of Parkinson’s disease (PD). The purpose of this study was to determine TNF-␣ levels in tear samples obtained from patients with PD and to analyze the relationship between TNF-␣ values and PD characteristics. Eighteen patients with PD and 17 healthy control subjects were included in the study. We examined the patient demographics, modified Hoehn and Yahr Staging Scale (mHY) stages, Unified Parkinson’s disease rating scale (UPDRS) II and III scores, Mini Mental State Examination (MMSE) scores, and the predominant symptoms. We measured TNF-␣ using the multiplex immunobead assay in unstimulated tear samples, and determined the Schirmer’s test and blink rate for each subject. Tear TNF-␣ values were significantly higher in patients with PD (196.9 ± 121.2 pg/ml) than in control subjects (110.7 ± 87.2 pg/ml; p = 0.02). We identified no relationship between tear TNF-␣ levels and age, sex, age at onset, PD duration, mHY stages, UPDRS II, UPDRS III, or MMSE scores. The higher TNF-␣ levels observed in the tears of patients with PD suggests neuroinflammation and TNF-␣ plays a role in the pathogenesis of PD. Tear TNF-␣ levels, however, were not related to the duration or severity of PD. Tears are a suitable method for measuring TNF-␣ levels, and can be used as a diagnostic measure to evaluate biomarkers in PD. © 2013 Published by Elsevier Ireland Ltd.
Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. The characteristic pathological signs of PD are a loss of dopaminergic neurons in the pars compacta region of the substantia nigra, and the presence of Lewy bodies, which are intracytoplasmic inclusions that are composed mainly of ␣-synuclein proteins. A series of complex events such as inflammation, mitochondrial dysfunction, oxidative and nitrosative stress, excitotoxicity, growth factor deficiencies,
∗ Corresponding author at: C¸i˘gdem mah. 1550/1 cad. 23/1 C¸ankaya, Ankara 06530, Turkey. Tel.: +90 5325778457; fax: +90 3123186690. E-mail addresses: [email protected], [email protected] (H. Güven).
accumulation of aberrant or misfolded proteins, and dysfunction of the ubiquitin-proteosome system occurs in the pathogenesis of PD, which leads to nigral cell death [16,19,26,34,37]. Microglia activation and neuroinflammation play an important role in the progressive degeneration of dopaminergic neurons in PD [16,27,34,37]. It was suggested that the neurotoxic effects of microglia-derived inflammatory mediators, particularly the proinflammatory cytokine tumor necrosis factor-alpha (TNF-␣), and their downstream signaling pathways might cause nigral dopaminergic neuron death. TNF-␣, which regulates maintenance of cellular vitality, gene expression, synaptic integrity, and ionic homeostasis, is a key mediator for cell death in chronic neuroinflammatory conditions [13,25]. In animal models, TNF-␣ and other pro-inflammatory cytokines can induce dopaminergic neuron loss [6,21]. Post-mortem
0304-3940/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.neulet.2013.08.019
Please cite this article in press as: S.S. C¸omo˘glu, et al., Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.08.019
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studies in patients with PD have identified TNF-immunoreactive glial cells in the substantia nigra [3], elevated TNF-␣ levels in nigrostriatal dopaminergic regions and cerebrospinal fluid, and have indicated TNF-␣ is released from activated glial cells in the substantia nigra [23,27]. Furthermore, caspase-1 and caspase-3 levels and the TNF-␣ receptor type-1 (TNF-R1), which initiates cell death mediated by caspase-1- and caspase-3-dependent pathways, were increased in nigrostriatal dopaminergic neurons in patients with PD, which implicates TNF-␣-induced signaling pathways in apoptotic cell death [24,27]. A significant correlation between putamen A2A adenosine receptor density and plasma TNF-␣ levels also was suggested in patients with PD, where TNF-␣ was thought to induce overexpression of A2A adenosine receptors [35]. Findings from genetic studies further support a TNF-␣ role in PD pathogenesis. Promoter region polymorphisms (−1031 and −308) on the TNF gene were associated with an increased risk of PD [36,38]. Moreover, elevated serum TNF-␣ levels have been observed in patients with PD [4,10,31,35]. TNF-␣ is a potential target for therapeutic interventions in degenerative diseases with chronic neuroinflammation such as PD, Alzheimer’s disease, and amyotrophic lateral sclerosis [13,34]. In experimental PD models, genetic ablation, and receptor deletion of TNF-␣, or agents inhibiting the synthesis and release of TNF-␣, provided protection to nigrostriatal dopaminergic neurons [12,32]. Although TNF-␣ has been measured in post-mortem brains, only cerebrospinal fluid and serum have been examined in patients with PD so far. TNF-␣ levels have not been investigated in tears, which offers a potential diagnostic marker in PD. In the present study, we aimed to determine TNF-␣ levels in the tears of patients with PD and then analyze the relationship between these TNF-␣ values and PD characteristics.
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2. Materials and methods
Both patients and the control subjects underwent a 15 min video recording from which blink rate was calculated by counting the number of blinks per minute.
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2.1. Subjects
2.5. Schirmer test A standard Schirmer filter paper strip (Tear Flo Sterile Strips, Rose Stone Enterprises, CA, USA) was inserted into the inferior lid margin 2–3 mm from the lateral canthus, without using a topical anesthetic agent. After 5 min, the moist part of the strip was measured starting from the edge of the eyelid. Tear collection, blink rate measurement, the Schirmer test, and a complete ophthalmologic examination, which included visual acuity, intraocular pressure measurement, and a detailed anterior segment and fundus examination, were performed by a single physician that was masked to the subject’s group.
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Eighteen patients with idiopathic PD and 17 healthy volunteers were selected as potential study participants. All potential participants underwent physical and neurological examinations, laboratory tests, and medical history evaluation. Patients were evaluated according to the UK Parkinson’s Disease Society Brain Bank Clinical Diagnostic Criteria to confirm the idiopathic PD diagnosis [15]. Each patient’s demographic information, including the age at disease onset and PD duration, were recorded. In addition, we determined for each patient the modified Hoehn and Yahr staging scale (mHY); unified Parkinson’s disease rating scale (UPDRS) subscores for daily life activities (UPDRS II) and motor examination (UPDRS III), mini mental state examination (MMSE) scores, and the predominant symptoms (tremor, bradykinesia, or tremor + bradykinesia). Patients continued to take anti-parkinsonian drugs other than anticholinergics during the study.
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All study participants were examined for exclusion criteria related to either systemic or ocular causes. Systemic exclusion criteria included the following: inflammatory, autoimmune, hematologic, or neoplastic disease; diabetes mellitus, thyroid disease, alcohol addiction, acute or chronic infection; history of hepatic or renal failure, myocardial infarction, cranial trauma and surgery within the last 6 months; abnormal white blood cell counts; use of diuretic, anti-inflammatory, antineoplastic, corticosteroid, immunosuppressive, antidepressant, or anxiolytic drugs within the last 2 months; use of anticholinergic drugs within the last 7 days. Ocular causes were an active ocular infection or signs of
inflammation, ocular allergy, use of ophthalmic drops or contact lenses, or ocular surgery within the last 3 months. The study was carried out according to the Helsinki Declaration and was approved by the Institutional Ethics Committee. All patients participating in the study provided informed consent. 2.3. Tear collection and multiplex immunobead assay analysis Unstimulated tear samples were collected non-traumatically from the external canthus of open eyes with a polyvinyl acetal sponge (Merocel, Beaver Visitec International, Waltham, MA, USA), which minimized irritation of the ocular surface and lid margin. Anesthetic drops were not used during tear collection. Tear samples were stored at −80 ◦ C until assayed. TNF-␣ measurements were performed with a Luminex 200 system (Luminex Corp., Austin, TX, USA) using the multiplex immunobead assay (Milliplex Map Kit, Millipore, USA). Each sample was diluted 1:5 times with assay buffer before analysis. For the analysis, 25 L of sample was incubated with antibody-coated magnetic beads for 2 h at room temperature. Washed beads were further incubated with detection antibody, followed by streptavidin-phycoerythrin incubation. Standard curves for known concentrations of human cytokine standards were used to convert fluorescence units to cytokine concentration (pg/ml). The samples were carried out together in the same experiment. Intra-assay %CV was 2.6 for TNF-␣. The same physician masked to the patient or control group performed the multiplex analysis. 2.4. Blink rate
2.6. Statistical analysis All statistical data analyses were performed with SPSS Statistics (version 19, IBM, Chicago, IL, USA). We characterized the demographic and clinical data for study participants by calculating the mean values and standard deviations for continuous variables, and calculating the incidence range and percentage values for categorical variables. For the patient group, we performed a Pearson correlation analysis to determine the relationship between TNF-␣ values and age, PD duration, age at onset of PD, UPDRS II, UPDRS III, MMSE scores, and mHY stages. In addition, the non-parametric Kruskal–Wallis test was used to examine the relationship between TNF-␣ values and the predominant symptom type. TNF-␣ values were compared between the patient and control groups using an independent sampling t-test, whereas the Mann–Whitney U test was used for comparisons between men and women because these data were not normally distributed. Statistical analyses were considered significant when p < 0.05.
Please cite this article in press as: S.S. C¸omo˘glu, et al., Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.08.019
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Table 1 Demographic and clinical characteristics of Parkinson’s disease patients. PD (n = 18) Age (years)a Gender (female/male)b Age of PD onset (years)a Duration of disease (years)a MMSE scorea mHY stagea UPDRS II scorea UPDRS III scorea
68.2 ± 10.7 5/13 (28/72) 60.3 ± 16.3 7.9 ± 7.5 26.9 ± 2.5 2.2 ± 0.9 7.6 ± 4.0 15 ± 5.2
Predominant symptom Tremorb Bradykinesiab Tremor + Bradykinesiab
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Abbreviations. PD, Parkinson’s disease; TNF-␣, tumor necrosis factor-alpha; MMSE, mini mental state examination; mHY, modified Hoehn and Yahr staging scale; UPDRS, unified Parkinson’s disease rating scale. a Data reported as mean ± SD. b Data reported as number (%).
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3. Results All study participants met the inclusion criteria and were included in the study. In total, 18 patients with PD (13 men, 5 women) and 17 healthy control subjects (11 men, 6 women) were included in the study. The mean age was 68.2 ± 10.7 (range, 41–87) years for the patient group and 62.9 ± 8.8 (37–76) years for the control group. MMSE scores were 26.9 ± 2.5 for the patient group and 27.17 ± 1.4 for the control group. There were no statistically significant differences between patient and control groups in terms of age, gender, MMSE scores and education level. The demographic and clinical characteristics of patients with PD are shown in Table 1. As Fig. 1 indicates, tear TNF-␣ values were significantly higher in patients with PD (196.9 ± 121.3 pg/ml) than in control subjects (110.7 ± 87.2 pg/ml, p = 0.02). Other comparisons between the PD and control groups revealed blink rate (number/min) was significantly lower in the PD group (5.6 ± 2.4) than in the control group (20.1 ± 3.0, p < 0.001). Schirmer-I test (mm/5 min) values also were significantly lower in the PD group (4.3 ± 1.8 mm) than in the control group (9.4 ± 3.0 mm, p < 0.001). Within the PD group, tear TNF-␣ values were greater in men (218.9 ± 126.7 pg/ml) than in women (139.7 ± 92.9 pg/ml), though this difference was not significant (p = 0.4). The mean tear TNF-␣ values were 296.3 ± 151.6 pg/ml when the predominant symptom was bradykinesia, which was greater than those observed with tremor (185.5 ± 110.9 pg/ml) and tremor + bradykinesia (151.6 ± 93.6 pg/ml). These differences in mean TNF-␣ values, however, were statistically insignificant (p = 0.2). Finally, we found no evidence for a relationship between tear TNF-␣ values and age, age at PD onset, PD duration, mHY stages,
Fig. 1. Tear tumor necrosis factor-alpha (TNF-␣) values of Parkinson’s disease (PD) and control groups.
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Table 2 Relationship between tear TNF-␣ levels and demographic and clinical characteristics in Parkinson’s disease patients. TNF-␣ level ra Age (years) Age of PD onset (years) Duration of disease (years) mHY stage UPDRS II score UPDRS III score MMSE score
0.239 0.052 −0.022 0.175 −0.041 0.093 −0.248
p 0.339 0.839 0.931 0.486 0.872 0.715 0.321
Abbreviations. TNF-␣, tumor necrosis factor-alpha; PD, Parkinson’s disease; MMSE, mini mental state examination; mHY, modified Hoehn and Yahr staging scale; UPDRS, unified Parkinson’s disease rating scale. a r, Pearson’s correlation coefficient.
UPDRS II, UPDRS III, or MMSE scores. Although we observed positive correlations between tear TNF-␣ values and age or mHY stages, and a negative correlation between tear TNF-␣ values and MMSE scores, these correlations were not statistically significant (Table 2). 4. Discussion We observed in the present study that, tear TNF-␣ levels were higher in patients with PD than in control subjects. This finding further implicates neuroinflammation and TNF-␣ in disease pathogenesis, and indicates that tears can be used to evaluate TNF-␣ levels, particularly in PD. TNF-␣, a potent pro-inflammatory cytokine, has a promoting role in neuroinflammation mediated progressive degeneration of dopaminergic neurons in PD [13,25]. Furthermore, TNF-␣ induced signaling pathway may play an important role in apoptotic cell death in PD [24,27]. It was found that the level of TNF-␣ was significantly increased in the lumbar cerebrospinal fluid and in the nigrostriatal region of the brain in PD as compared to other cytokines such as interleukin-1beta (IL-1), IL-2, IL-4, IL-6, transforming growth factor-alpha (TGF-␣), and TGF-1 [23,27]. There is a growing evidence of the role of TNF-␣ in the pathogenesis of PD based on data from many clinical studies and animal models [4,6,10,21,31,35]. Although TNF-␣ measurements have been carried out in postmortem brains, in cerebrospinal fluid, and in blood samples of patients with PD, TNF-␣ levels have not been investigated in tears. We therefore chose to examine the presence of TNF-␣ levels in tears for several reasons. First, with idiopathic PD, ␣-synuclein pathology can occur in many tissues, particularly in the spinal cord and autonomic nervous system, before neurons are lost in the pars compacta region of the substantia nigra [2,7,30]. The lacrimal glands are directly influenced by the autonomic nervous system, and receive both parasympathetic and sympathetic innervation. In PD, patients can demonstrate decreased tear secretion that is associated with autonomic dysfunction in the lacrimal glands [1,18,33]. Secondly, conjunctival epithelial cells participate in immune reactions by expressing surface antigens, such as adhesion molecules, and secreting cytokines and chemokines [11,17]. Conjunctival epithelial cells also can be affected by the inflammatory pathology or reflect inflammation in PD. Therefore, tear TNF-␣ levels can be a marker of inflammation in PD. Finally, ␣-synuclein and DJ-1 proteins can be detected in the saliva of patients with PD, which can be a potential diagnostic marker for PD [8]. Furthermore, neuropathologic examinations have revealed the submandibular gland is affected by synucleinopathy [2,7]. Similar to the submandibular gland, the lacrimal gland can be affected in PD. Aging is considered the greatest risk factor for PD [19]. New findings have shown the aging process has a priming effect on microglia, and excessive pro-inflammatory cytokines may be released from
Please cite this article in press as: S.S. C¸omo˘glu, et al., Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.08.019
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microglia through a triggering stimulus [9]. Furthermore, TNF-␣ was found to be higher in centenarians compared with younger individuals, and this result was suggested to point to an age-related immune activation [5]. Although a positive correlation was found in the present study between patient age and tear TNF-␣ levels, this finding was statistically insignificant. An absence of a relationship between TNF-␣ levels and aging indicate that an increased TNF-␣ is associated with PD, and is independent from the aging process. We found no evidence that tear TNF-␣ levels were associated with age at PD onset or disease duration. This suggests TNF-␣ may be an indicator for disease pathogenesis, but not for disease duration or progression. It is known that neurodegeneration in PD begins in the premotor period, years before the characteristic symptoms of PD first appear [14]. Our inability to find an association between TNF-␣ and disease duration raises the question of whether TNF-␣ measurements are useful for determining PD risk before the onset of symptoms. No significant correlation was found between tear TNF-␣ levels and UPDRS III scores or mHY disease stage. Such a result might indicate TNF-␣ levels are unrelated to the motor symptoms of PD, and do not reflect the severity of PD. A correlation between serum TNF-␣ levels and UPDRS motor scores and Hoehn–Yahr stages has been observed in patients with PD [31,35], but other investigations have failed to identify correlations between motor dysfunction and TNF-␣ levels [10]. The lack of correlation between TNF-␣ levels and the severity of motor symptoms in PD in the present study does not conclusively determine whether TNF-␣ is associated with the severity of PD disease, because motor findings reflect only the involvement of nigrostriatal dopaminergic system. Furthermore, all patients in the present study were under dopaminergic treatment that may alleviate motor signs and symptoms. We identified a trend in which TNF-␣ values increased as MMSE scores decreased, though this trend was statistically insignificant. However, absence of advanced cognitive impairment in our patients might have influenced our findings. Although one study revealed a relationship between serum TNF-␣ levels and cognitive dysfunction in patients with PD [22], another study found no relationship between TNF-␣ levels and cognitive dysfunction [10]. In addition to elevated tear TNF-␣ levels, dry eye was more common in patients with PD than in control subjects. It is possible that decreased blink rate in PD may cause evaporative-type dry eye, and consequently tear TNF-␣ may increase. In chronic inflammatory diseases of the ocular surface, such as dry eye, tear cytokine and chemokine levels have been reported to increase [20,29]. Nevertheless, elevation in tear cytokines is rather seen in more severe dry eye disease associated with Sjögren syndrome [28,29]. Marked elevation in TNF-␣ in patients with PD compared with the control group supports our hypothesis that inflammatory events associated with PD may influence TNF-␣ elevation. 5. Conclusions The present study showed that TNF-␣ was elevated in the tears of patients with PD, but TNF-␣ levels were not associated with disease duration and did not reflect severity of motor findings. Furthermore, tears are a potential source for evaluating biomarkers in PD. The findings from this preliminary study should be examined further with larger patient groups. Future studies should examine whether TNF-␣ can predict PD risk or provide early diagnosis, and determine treatments that target TNF-␣ in neurodegenerative disorders such as PD. Conflict of interest None
References [1] H. Bagheri, M. Berlan, J.M. Senard, O. Rascol, J.L. Montastruc, Lacrimation in Parkinson’s disease, Clin. Neuropharmacol. 17 (1994) 89–91. [2] T.G. Beach, C.H. Adler, L.I. Sue, L. Vedders, L. Lue, IiiC.L. White, H. Akiyama, J.N. Caviness, H.A. Shill, M.N. Sabbagh, WalkerF D.G., Arizona Parkinson’s disease consortium, multi-organ distribution of phosphorylated alpha-synuclein histopathology in subjects with Lewy body disorders, Acta Neuropathol. 119 (2010) 689–702. [3] G. Boka, P. Anglade, D. Wallach, F. Javoy-Agid, Y. Agid, E.C. Hirsch, Imminocytochemical analysis of tumor necrosis factor and its receptors in Parkinson’s disease, Neurosci. Lett. 172 (1994) 151–154. [4] B. Brodacki, J. Staszewski, E. Toczylowska, E. Kozłowska, N. Drela, M. Chalimoniuk, A. Stepien, Serum interleukin (IL-2, IL-10, IL-6, IL-4), TNF-alpha, and INF-gamma concentrations are elevated in patients with atypical and idiopathic parkinsonism, Neurosci. Lett. 441 (2008) 158–162. [5] H. Bruunsgaard, K. Andersen-Ranberg, B. Jeune, A.N. Pedersen, P. Skinhøj, B.K. Pedersen, A high plasma concentration of TNF-alpha is associated with dementia in centenarians, J. Gerontol. A. Biol. Sci. Med. Sci. 54 (1999) M357–M364. [6] P.M. Carvey, E.Y. Chen, J.W. Lipton, C.W. Tong, Q.A. Chang, Z.D. Ling, Intraparenchymal injection of tumor necrosis factor-alpha and interleukin 1-beta produces dopamine neuron loss in the rat, J. Neural Transm. 112 (2005) 601–612. [7] K. Del Tredici, C.H. Hawkes, E. Ghebremedhin, H. Braak, Lewy pathology in the submandibular gland of individuals with incidental Lewy body disease and sporadic Parkinson’s disease, Acta Neuropathol. 119 (2010) 703–713. [8] I. Devic, H. Hwang, J.S. Edgar, K. Izutsu, R. Presland, C. Pan, D.R. Goodlett, Y. Wang, J. Armaly, V. Tumas, C.P. Zabetian, J.B. Leverenz, M. Shi, J. Zhang, Salivary ␣-synuclein and DJ-1: potential biomarkers for Parkinson’s disease, Brain 134 (Pt 7) (2011) e178. [9] R.N. Dilger, R.W. Johnson, Aging, microglial cell priming, and the discordant central inflammatory response to signals from the peripheral immune system, J. Leukoc. Biol. 84 (2008) 932–939. [10] M. Dufek, M. Hamanová, J. Lokaj, D. Goldemund, I. Rektorová, Z. Michálková, K. Sheardová, I. Rektor, Serum inflammatory biomarkers in Parkinson’s disease, Parkinsonism Relat. Disord. 15 (2009) 318–320. [11] A. Enríquez-de-Salamanca, V. Calder, J. Gao, G. Galatowicz, C. García-Vázquez, I. Fernández, M.E. Stern, Y. Diebold, M. Calonge, Cytokine responses by conjunctival epithelial cells: an in vitro model of ocular inflammation, Cytokine 44 (2008) 160–167. [12] B. Ferger, A. Leng, A. Mura, B. Hengerer, J. Feldon, Genetic ablation of tumor necrosis factor-alpha (TNF-alpha) and pharmacological inhibition of TNFsynthesis attenuates MPTP toxicity in mouse striatum, J. Neurochem. 89 (2004) 822–833. [13] K.A. Frankola, N.H. Greig, W. Luo, D. Tweedie, Targeting TNF-␣ to elucidate and ameliorate neuroinflammation in neurodegenerative diseases, CNS Neurol. Disord. Drug Targets 10 (2011) 391–403. [14] C. Gaig, E. Tolosa, When does Parkinson’s disease begin? Mov Disord. 24 (Suppl 2) (2009) S656–S664. [15] A.J. Hughes, S.E. Daniel, L. Kilford, A.J. Lees, Accuracy of clinical diagnosis of idiopathic Parkinson’s disease: a clinico-pathological study of 100 cases, J. Neurol. Neurosurg. Psychiat. 55 (1992) 181–184. [16] S. Hunot, E.C. Hirsch, Neuroinflammatory processes in Parkinson’s disease, Ann. Neurol. 53 (Suppl 3) (2003) S49–S58 (discussion S58–S60). [17] M. Irkec¸, B. Bozkurt, Epithelial cells in ocular allergy, Curr. Allergy Asthma Rep. 3 (2003) 352–357. [18] O.Y. Kwon, S.H. Kim, J.H. Kim, M.H. Kim, M.K. Ko, Schrimer test in Parkinson’s disease, J. Korean Med. Sci. 9 (1994) 239–242. [19] A.J. Lees, J. Hardy, T. Revesz, Parkinson’s disease, Lancet 373 (2009) 2055–2066. [20] M.L. Massingale, X. Li, M. Vallabhajosyula, D. Chen, Y. Wei, P.A. Asbell, Analysis of inflammatory cytokines in the tears of dry eye patients, Cornea 28 (2009) 1023–1027. [21] S.O. McGuire, Z.D. Ling, J.W. Lipton, C.E. Sortwell, T.J. Collier, P.M. Carvey, Tumor necrosis factor alpha is toxic to embryonic mesencephalic dopamine neurons, Exp. Neurol. 169 (2001) 219–230. [22] M. Menza, R.D. Dobkin, H. Marin, M.H. Mark, M. Gara, K. Bienfait, A. Dicke, A. Kusnekov, The role of inflammatory cytokines in cognition and other non-motor symptoms of Parkinson’s disease, Psychosomatics 51 (2010) 474–479. [23] M. Mogi, M. Harada, P. Riederer, H. Narabayashi, K. Fujita, T. Nagatsu, Tumor necrosis factor-alpha (TNF-alpha) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients, Neurosci. Lett. 165 (1994) 208–210. [24] M. Mogi, A. Togari, T. Kondo, Y. Mizuno, O. Komure, S. Kuno, H. Ichinose, T. Nagatsu, Caspase activities and tumor necrosis factor receptor R1 (p55) level are elevated in the substantia nigra from parkinsonian brain, J. Neural Transm. 107 (2000) 335–341. [25] S.L. Montgomery, W.J. Bowers, Tumor necrosis factor-alpha and the roles it plays in homeostatic and degenerative processes within the central nervous system, J. Neuroimmune Pharmacol. 7 (2012) 42–59. [26] D.J. Moore, A.B. West, V.L. Dawson, T.M. Dawson, Molecular pathophysiology of Parkinson’s disease, Annu. Rev. Neurosci. 28 (2005) 57–87. [27] T. Nagatsu, M. Sawada, Inflammatory process in Parkinson’s disease: role for cytokines, Curr. Pharm. Des. 11 (2005) 999–1016. [28] S. Narayanan, W.L. Miller, A.M. McDermott, Conjunctival cytokine expression in symptomatic moderate dry eye subjects, Invest. Ophthalmol. Vis. Sci. 47 (2006) 2445–2450.
Please cite this article in press as: S.S. C¸omo˘glu, et al., Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.08.019
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[29] S.C. Pflugfelder, D. Jones, Z. Ji, A. Afonso, D. Monroy, Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjögren’s syndrome keratoconjunctivitis sicca, Curr. Eye Res. 19 (1999) 201–211. [30] A. Probst, A. Bloch, M. Tolnay, New insights into the pathology of Parkinson’s disease: does the peripheral autonomic system become central? Eur. J. Neurol. 15 (Suppl 1) (2008) 1–4. [31] M. Reale, C. Iarlori, A. Thomas, D. Gambi, B. Perfetti, M. Di Nicola, M. Onofrj, Peripheral cytokines profile in Parkinson’s disease, Brain Behav. Immun. 23 (2009) 55–63. [32] K. Sriram, J.M. Matheson, S.A. Benkovic, D.B. Miller, M.I. Luster, J.P. O’Callaghan, Mice deficient in TNF receptors are protected against dopaminergic neurotoxicity: implications for Parkinson’s disease, FASEB J. 16 (2002) 1474–1476. [33] C. Tamer, I.M. Melek, T. Duman, H. Oksüz, Tear film tests in Parkinson’s disease patients, Ophthalmology 112 (2005) 1795.
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[34] M.G. Tansey, M.S. Goldberg, Neuroinflammation in Parkinson’s disease: its role in neuronal death and implications for therapeutic intervention, Neurobiol. Dis. 37 (2010) 510–518. [35] K. Varani, F. Vincenzi, A. Tosi, S. Gessi, I. Casetta, G. Granieri, P. Fazio, E. Leung, S. MacLennan, E. Granieri, P.A. Borea, A2A adenosine receptor overexpression and functionality, as well as TNF-alpha levels, correlate with motor symptoms in Parkinson’s disease, FASEB J. 24 (2010) 587–598. [36] A.D. Wahner, J.S. Sinsheimer, J.M. Bronstein, B. Ritz, Inflammatory cytokine gene polymorphisms and increased risk of Parkinson’s disease, Arch. Neurol. 64 (2007) 836–840. [37] P.S. Whitton, Inflammation as a causative factor in the aetiology of Parkinson’s disease, Br. J. Pharmacol. 150 (2007) 963–976. [38] Y.R. Wu, I.H. Feng, R.K. Lyu, K.H. Chang, Y.Y. Lin, H. Chan, F.J. Hu, G.J. Lee-Chen, C.M. Chen, Tumor necrosis factor-alpha promoter polymorphism is associated with the risk of Parkinson’s disease, Am. J. Med. Genet. B. Neuropsychiatr. Genet. 144B (2007) 300–304.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease
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Selim Selc¸uk C¸omo˘glu a , Hayat Güven a,∗ , Mutlu Acar b , Gülfer Öztürk c , Bilge Koc¸er a
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Ankara Dıs¸kapı Yıldırım Beyazıt Training and Research Hospital, Department of Neurology, Turkey Ankara Dıs¸kapı Yıldırım Beyazıt Training and Research Hospital, Department of Ophthalmology, Turkey Ankara Dıs¸kapı Yıldırım Beyazıt Training and Research Hospital, Department of Biochemistry, Turkey
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h i g h l i g h t s • TNF-␣ and other pro-inflammatory cytokines can induce dopaminergic neuron loss. • We aimed to determine TNF-␣ levels in the tears of patients with PD. • Tear TNF-␣ values were significantly higher in patients with PD than in control subjects.
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Article history: Received 12 June 2013 Received in revised form 7 August 2013 Accepted 12 August 2013
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Keywords: Tumor necrosis factor-alpha Neuroinflammation Parkinson’s disease Tear
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1. Introduction
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Neuroinflammatory events mediated by the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-␣) cause progressive neurodegeneration in dopaminergic neurons, and play an important role in the pathogenesis of Parkinson’s disease (PD). The purpose of this study was to determine TNF-␣ levels in tear samples obtained from patients with PD and to analyze the relationship between TNF-␣ values and PD characteristics. Eighteen patients with PD and 17 healthy control subjects were included in the study. We examined the patient demographics, modified Hoehn and Yahr Staging Scale (mHY) stages, Unified Parkinson’s disease rating scale (UPDRS) II and III scores, Mini Mental State Examination (MMSE) scores, and the predominant symptoms. We measured TNF-␣ using the multiplex immunobead assay in unstimulated tear samples, and determined the Schirmer’s test and blink rate for each subject. Tear TNF-␣ values were significantly higher in patients with PD (196.9 ± 121.2 pg/ml) than in control subjects (110.7 ± 87.2 pg/ml; p = 0.02). We identified no relationship between tear TNF-␣ levels and age, sex, age at onset, PD duration, mHY stages, UPDRS II, UPDRS III, or MMSE scores. The higher TNF-␣ levels observed in the tears of patients with PD suggests neuroinflammation and TNF-␣ plays a role in the pathogenesis of PD. Tear TNF-␣ levels, however, were not related to the duration or severity of PD. Tears are a suitable method for measuring TNF-␣ levels, and can be used as a diagnostic measure to evaluate biomarkers in PD. © 2013 Published by Elsevier Ireland Ltd.
Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. The characteristic pathological signs of PD are a loss of dopaminergic neurons in the pars compacta region of the substantia nigra, and the presence of Lewy bodies, which are intracytoplasmic inclusions that are composed mainly of ␣-synuclein proteins. A series of complex events such as inflammation, mitochondrial dysfunction, oxidative and nitrosative stress, excitotoxicity, growth factor deficiencies,
∗ Corresponding author at: C¸i˘gdem mah. 1550/1 cad. 23/1 C¸ankaya, Ankara 06530, Turkey. Tel.: +90 5325778457; fax: +90 3123186690. E-mail addresses: [email protected], [email protected] (H. Güven).
accumulation of aberrant or misfolded proteins, and dysfunction of the ubiquitin-proteosome system occurs in the pathogenesis of PD, which leads to nigral cell death [16,19,26,34,37]. Microglia activation and neuroinflammation play an important role in the progressive degeneration of dopaminergic neurons in PD [16,27,34,37]. It was suggested that the neurotoxic effects of microglia-derived inflammatory mediators, particularly the proinflammatory cytokine tumor necrosis factor-alpha (TNF-␣), and their downstream signaling pathways might cause nigral dopaminergic neuron death. TNF-␣, which regulates maintenance of cellular vitality, gene expression, synaptic integrity, and ionic homeostasis, is a key mediator for cell death in chronic neuroinflammatory conditions [13,25]. In animal models, TNF-␣ and other pro-inflammatory cytokines can induce dopaminergic neuron loss [6,21]. Post-mortem
0304-3940/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.neulet.2013.08.019
Please cite this article in press as: S.S. C¸omo˘glu, et al., Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.08.019
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studies in patients with PD have identified TNF-immunoreactive glial cells in the substantia nigra [3], elevated TNF-␣ levels in nigrostriatal dopaminergic regions and cerebrospinal fluid, and have indicated TNF-␣ is released from activated glial cells in the substantia nigra [23,27]. Furthermore, caspase-1 and caspase-3 levels and the TNF-␣ receptor type-1 (TNF-R1), which initiates cell death mediated by caspase-1- and caspase-3-dependent pathways, were increased in nigrostriatal dopaminergic neurons in patients with PD, which implicates TNF-␣-induced signaling pathways in apoptotic cell death [24,27]. A significant correlation between putamen A2A adenosine receptor density and plasma TNF-␣ levels also was suggested in patients with PD, where TNF-␣ was thought to induce overexpression of A2A adenosine receptors [35]. Findings from genetic studies further support a TNF-␣ role in PD pathogenesis. Promoter region polymorphisms (−1031 and −308) on the TNF gene were associated with an increased risk of PD [36,38]. Moreover, elevated serum TNF-␣ levels have been observed in patients with PD [4,10,31,35]. TNF-␣ is a potential target for therapeutic interventions in degenerative diseases with chronic neuroinflammation such as PD, Alzheimer’s disease, and amyotrophic lateral sclerosis [13,34]. In experimental PD models, genetic ablation, and receptor deletion of TNF-␣, or agents inhibiting the synthesis and release of TNF-␣, provided protection to nigrostriatal dopaminergic neurons [12,32]. Although TNF-␣ has been measured in post-mortem brains, only cerebrospinal fluid and serum have been examined in patients with PD so far. TNF-␣ levels have not been investigated in tears, which offers a potential diagnostic marker in PD. In the present study, we aimed to determine TNF-␣ levels in the tears of patients with PD and then analyze the relationship between these TNF-␣ values and PD characteristics.
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2. Materials and methods
Both patients and the control subjects underwent a 15 min video recording from which blink rate was calculated by counting the number of blinks per minute.
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2.1. Subjects
2.5. Schirmer test A standard Schirmer filter paper strip (Tear Flo Sterile Strips, Rose Stone Enterprises, CA, USA) was inserted into the inferior lid margin 2–3 mm from the lateral canthus, without using a topical anesthetic agent. After 5 min, the moist part of the strip was measured starting from the edge of the eyelid. Tear collection, blink rate measurement, the Schirmer test, and a complete ophthalmologic examination, which included visual acuity, intraocular pressure measurement, and a detailed anterior segment and fundus examination, were performed by a single physician that was masked to the subject’s group.
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Eighteen patients with idiopathic PD and 17 healthy volunteers were selected as potential study participants. All potential participants underwent physical and neurological examinations, laboratory tests, and medical history evaluation. Patients were evaluated according to the UK Parkinson’s Disease Society Brain Bank Clinical Diagnostic Criteria to confirm the idiopathic PD diagnosis [15]. Each patient’s demographic information, including the age at disease onset and PD duration, were recorded. In addition, we determined for each patient the modified Hoehn and Yahr staging scale (mHY); unified Parkinson’s disease rating scale (UPDRS) subscores for daily life activities (UPDRS II) and motor examination (UPDRS III), mini mental state examination (MMSE) scores, and the predominant symptoms (tremor, bradykinesia, or tremor + bradykinesia). Patients continued to take anti-parkinsonian drugs other than anticholinergics during the study.
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All study participants were examined for exclusion criteria related to either systemic or ocular causes. Systemic exclusion criteria included the following: inflammatory, autoimmune, hematologic, or neoplastic disease; diabetes mellitus, thyroid disease, alcohol addiction, acute or chronic infection; history of hepatic or renal failure, myocardial infarction, cranial trauma and surgery within the last 6 months; abnormal white blood cell counts; use of diuretic, anti-inflammatory, antineoplastic, corticosteroid, immunosuppressive, antidepressant, or anxiolytic drugs within the last 2 months; use of anticholinergic drugs within the last 7 days. Ocular causes were an active ocular infection or signs of
inflammation, ocular allergy, use of ophthalmic drops or contact lenses, or ocular surgery within the last 3 months. The study was carried out according to the Helsinki Declaration and was approved by the Institutional Ethics Committee. All patients participating in the study provided informed consent. 2.3. Tear collection and multiplex immunobead assay analysis Unstimulated tear samples were collected non-traumatically from the external canthus of open eyes with a polyvinyl acetal sponge (Merocel, Beaver Visitec International, Waltham, MA, USA), which minimized irritation of the ocular surface and lid margin. Anesthetic drops were not used during tear collection. Tear samples were stored at −80 ◦ C until assayed. TNF-␣ measurements were performed with a Luminex 200 system (Luminex Corp., Austin, TX, USA) using the multiplex immunobead assay (Milliplex Map Kit, Millipore, USA). Each sample was diluted 1:5 times with assay buffer before analysis. For the analysis, 25 L of sample was incubated with antibody-coated magnetic beads for 2 h at room temperature. Washed beads were further incubated with detection antibody, followed by streptavidin-phycoerythrin incubation. Standard curves for known concentrations of human cytokine standards were used to convert fluorescence units to cytokine concentration (pg/ml). The samples were carried out together in the same experiment. Intra-assay %CV was 2.6 for TNF-␣. The same physician masked to the patient or control group performed the multiplex analysis. 2.4. Blink rate
2.6. Statistical analysis All statistical data analyses were performed with SPSS Statistics (version 19, IBM, Chicago, IL, USA). We characterized the demographic and clinical data for study participants by calculating the mean values and standard deviations for continuous variables, and calculating the incidence range and percentage values for categorical variables. For the patient group, we performed a Pearson correlation analysis to determine the relationship between TNF-␣ values and age, PD duration, age at onset of PD, UPDRS II, UPDRS III, MMSE scores, and mHY stages. In addition, the non-parametric Kruskal–Wallis test was used to examine the relationship between TNF-␣ values and the predominant symptom type. TNF-␣ values were compared between the patient and control groups using an independent sampling t-test, whereas the Mann–Whitney U test was used for comparisons between men and women because these data were not normally distributed. Statistical analyses were considered significant when p < 0.05.
Please cite this article in press as: S.S. C¸omo˘glu, et al., Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.08.019
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Table 1 Demographic and clinical characteristics of Parkinson’s disease patients. PD (n = 18) Age (years)a Gender (female/male)b Age of PD onset (years)a Duration of disease (years)a MMSE scorea mHY stagea UPDRS II scorea UPDRS III scorea
68.2 ± 10.7 5/13 (28/72) 60.3 ± 16.3 7.9 ± 7.5 26.9 ± 2.5 2.2 ± 0.9 7.6 ± 4.0 15 ± 5.2
Predominant symptom Tremorb Bradykinesiab Tremor + Bradykinesiab
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Abbreviations. PD, Parkinson’s disease; TNF-␣, tumor necrosis factor-alpha; MMSE, mini mental state examination; mHY, modified Hoehn and Yahr staging scale; UPDRS, unified Parkinson’s disease rating scale. a Data reported as mean ± SD. b Data reported as number (%).
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3. Results All study participants met the inclusion criteria and were included in the study. In total, 18 patients with PD (13 men, 5 women) and 17 healthy control subjects (11 men, 6 women) were included in the study. The mean age was 68.2 ± 10.7 (range, 41–87) years for the patient group and 62.9 ± 8.8 (37–76) years for the control group. MMSE scores were 26.9 ± 2.5 for the patient group and 27.17 ± 1.4 for the control group. There were no statistically significant differences between patient and control groups in terms of age, gender, MMSE scores and education level. The demographic and clinical characteristics of patients with PD are shown in Table 1. As Fig. 1 indicates, tear TNF-␣ values were significantly higher in patients with PD (196.9 ± 121.3 pg/ml) than in control subjects (110.7 ± 87.2 pg/ml, p = 0.02). Other comparisons between the PD and control groups revealed blink rate (number/min) was significantly lower in the PD group (5.6 ± 2.4) than in the control group (20.1 ± 3.0, p < 0.001). Schirmer-I test (mm/5 min) values also were significantly lower in the PD group (4.3 ± 1.8 mm) than in the control group (9.4 ± 3.0 mm, p < 0.001). Within the PD group, tear TNF-␣ values were greater in men (218.9 ± 126.7 pg/ml) than in women (139.7 ± 92.9 pg/ml), though this difference was not significant (p = 0.4). The mean tear TNF-␣ values were 296.3 ± 151.6 pg/ml when the predominant symptom was bradykinesia, which was greater than those observed with tremor (185.5 ± 110.9 pg/ml) and tremor + bradykinesia (151.6 ± 93.6 pg/ml). These differences in mean TNF-␣ values, however, were statistically insignificant (p = 0.2). Finally, we found no evidence for a relationship between tear TNF-␣ values and age, age at PD onset, PD duration, mHY stages,
Fig. 1. Tear tumor necrosis factor-alpha (TNF-␣) values of Parkinson’s disease (PD) and control groups.
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Table 2 Relationship between tear TNF-␣ levels and demographic and clinical characteristics in Parkinson’s disease patients. TNF-␣ level ra Age (years) Age of PD onset (years) Duration of disease (years) mHY stage UPDRS II score UPDRS III score MMSE score
0.239 0.052 −0.022 0.175 −0.041 0.093 −0.248
p 0.339 0.839 0.931 0.486 0.872 0.715 0.321
Abbreviations. TNF-␣, tumor necrosis factor-alpha; PD, Parkinson’s disease; MMSE, mini mental state examination; mHY, modified Hoehn and Yahr staging scale; UPDRS, unified Parkinson’s disease rating scale. a r, Pearson’s correlation coefficient.
UPDRS II, UPDRS III, or MMSE scores. Although we observed positive correlations between tear TNF-␣ values and age or mHY stages, and a negative correlation between tear TNF-␣ values and MMSE scores, these correlations were not statistically significant (Table 2). 4. Discussion We observed in the present study that, tear TNF-␣ levels were higher in patients with PD than in control subjects. This finding further implicates neuroinflammation and TNF-␣ in disease pathogenesis, and indicates that tears can be used to evaluate TNF-␣ levels, particularly in PD. TNF-␣, a potent pro-inflammatory cytokine, has a promoting role in neuroinflammation mediated progressive degeneration of dopaminergic neurons in PD [13,25]. Furthermore, TNF-␣ induced signaling pathway may play an important role in apoptotic cell death in PD [24,27]. It was found that the level of TNF-␣ was significantly increased in the lumbar cerebrospinal fluid and in the nigrostriatal region of the brain in PD as compared to other cytokines such as interleukin-1beta (IL-1), IL-2, IL-4, IL-6, transforming growth factor-alpha (TGF-␣), and TGF-1 [23,27]. There is a growing evidence of the role of TNF-␣ in the pathogenesis of PD based on data from many clinical studies and animal models [4,6,10,21,31,35]. Although TNF-␣ measurements have been carried out in postmortem brains, in cerebrospinal fluid, and in blood samples of patients with PD, TNF-␣ levels have not been investigated in tears. We therefore chose to examine the presence of TNF-␣ levels in tears for several reasons. First, with idiopathic PD, ␣-synuclein pathology can occur in many tissues, particularly in the spinal cord and autonomic nervous system, before neurons are lost in the pars compacta region of the substantia nigra [2,7,30]. The lacrimal glands are directly influenced by the autonomic nervous system, and receive both parasympathetic and sympathetic innervation. In PD, patients can demonstrate decreased tear secretion that is associated with autonomic dysfunction in the lacrimal glands [1,18,33]. Secondly, conjunctival epithelial cells participate in immune reactions by expressing surface antigens, such as adhesion molecules, and secreting cytokines and chemokines [11,17]. Conjunctival epithelial cells also can be affected by the inflammatory pathology or reflect inflammation in PD. Therefore, tear TNF-␣ levels can be a marker of inflammation in PD. Finally, ␣-synuclein and DJ-1 proteins can be detected in the saliva of patients with PD, which can be a potential diagnostic marker for PD [8]. Furthermore, neuropathologic examinations have revealed the submandibular gland is affected by synucleinopathy [2,7]. Similar to the submandibular gland, the lacrimal gland can be affected in PD. Aging is considered the greatest risk factor for PD [19]. New findings have shown the aging process has a priming effect on microglia, and excessive pro-inflammatory cytokines may be released from
Please cite this article in press as: S.S. C¸omo˘glu, et al., Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.08.019
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microglia through a triggering stimulus [9]. Furthermore, TNF-␣ was found to be higher in centenarians compared with younger individuals, and this result was suggested to point to an age-related immune activation [5]. Although a positive correlation was found in the present study between patient age and tear TNF-␣ levels, this finding was statistically insignificant. An absence of a relationship between TNF-␣ levels and aging indicate that an increased TNF-␣ is associated with PD, and is independent from the aging process. We found no evidence that tear TNF-␣ levels were associated with age at PD onset or disease duration. This suggests TNF-␣ may be an indicator for disease pathogenesis, but not for disease duration or progression. It is known that neurodegeneration in PD begins in the premotor period, years before the characteristic symptoms of PD first appear [14]. Our inability to find an association between TNF-␣ and disease duration raises the question of whether TNF-␣ measurements are useful for determining PD risk before the onset of symptoms. No significant correlation was found between tear TNF-␣ levels and UPDRS III scores or mHY disease stage. Such a result might indicate TNF-␣ levels are unrelated to the motor symptoms of PD, and do not reflect the severity of PD. A correlation between serum TNF-␣ levels and UPDRS motor scores and Hoehn–Yahr stages has been observed in patients with PD [31,35], but other investigations have failed to identify correlations between motor dysfunction and TNF-␣ levels [10]. The lack of correlation between TNF-␣ levels and the severity of motor symptoms in PD in the present study does not conclusively determine whether TNF-␣ is associated with the severity of PD disease, because motor findings reflect only the involvement of nigrostriatal dopaminergic system. Furthermore, all patients in the present study were under dopaminergic treatment that may alleviate motor signs and symptoms. We identified a trend in which TNF-␣ values increased as MMSE scores decreased, though this trend was statistically insignificant. However, absence of advanced cognitive impairment in our patients might have influenced our findings. Although one study revealed a relationship between serum TNF-␣ levels and cognitive dysfunction in patients with PD [22], another study found no relationship between TNF-␣ levels and cognitive dysfunction [10]. In addition to elevated tear TNF-␣ levels, dry eye was more common in patients with PD than in control subjects. It is possible that decreased blink rate in PD may cause evaporative-type dry eye, and consequently tear TNF-␣ may increase. In chronic inflammatory diseases of the ocular surface, such as dry eye, tear cytokine and chemokine levels have been reported to increase [20,29]. Nevertheless, elevation in tear cytokines is rather seen in more severe dry eye disease associated with Sjögren syndrome [28,29]. Marked elevation in TNF-␣ in patients with PD compared with the control group supports our hypothesis that inflammatory events associated with PD may influence TNF-␣ elevation. 5. Conclusions The present study showed that TNF-␣ was elevated in the tears of patients with PD, but TNF-␣ levels were not associated with disease duration and did not reflect severity of motor findings. Furthermore, tears are a potential source for evaluating biomarkers in PD. The findings from this preliminary study should be examined further with larger patient groups. Future studies should examine whether TNF-␣ can predict PD risk or provide early diagnosis, and determine treatments that target TNF-␣ in neurodegenerative disorders such as PD. Conflict of interest None
References [1] H. Bagheri, M. Berlan, J.M. Senard, O. Rascol, J.L. Montastruc, Lacrimation in Parkinson’s disease, Clin. Neuropharmacol. 17 (1994) 89–91. [2] T.G. Beach, C.H. Adler, L.I. Sue, L. Vedders, L. Lue, IiiC.L. White, H. Akiyama, J.N. Caviness, H.A. Shill, M.N. Sabbagh, WalkerF D.G., Arizona Parkinson’s disease consortium, multi-organ distribution of phosphorylated alpha-synuclein histopathology in subjects with Lewy body disorders, Acta Neuropathol. 119 (2010) 689–702. [3] G. Boka, P. Anglade, D. Wallach, F. Javoy-Agid, Y. Agid, E.C. Hirsch, Imminocytochemical analysis of tumor necrosis factor and its receptors in Parkinson’s disease, Neurosci. Lett. 172 (1994) 151–154. [4] B. Brodacki, J. Staszewski, E. Toczylowska, E. Kozłowska, N. Drela, M. Chalimoniuk, A. Stepien, Serum interleukin (IL-2, IL-10, IL-6, IL-4), TNF-alpha, and INF-gamma concentrations are elevated in patients with atypical and idiopathic parkinsonism, Neurosci. Lett. 441 (2008) 158–162. [5] H. Bruunsgaard, K. Andersen-Ranberg, B. Jeune, A.N. Pedersen, P. Skinhøj, B.K. Pedersen, A high plasma concentration of TNF-alpha is associated with dementia in centenarians, J. Gerontol. A. Biol. Sci. Med. Sci. 54 (1999) M357–M364. [6] P.M. Carvey, E.Y. Chen, J.W. Lipton, C.W. Tong, Q.A. Chang, Z.D. Ling, Intraparenchymal injection of tumor necrosis factor-alpha and interleukin 1-beta produces dopamine neuron loss in the rat, J. Neural Transm. 112 (2005) 601–612. [7] K. Del Tredici, C.H. Hawkes, E. Ghebremedhin, H. Braak, Lewy pathology in the submandibular gland of individuals with incidental Lewy body disease and sporadic Parkinson’s disease, Acta Neuropathol. 119 (2010) 703–713. [8] I. Devic, H. Hwang, J.S. Edgar, K. Izutsu, R. Presland, C. Pan, D.R. Goodlett, Y. Wang, J. Armaly, V. Tumas, C.P. Zabetian, J.B. Leverenz, M. Shi, J. Zhang, Salivary ␣-synuclein and DJ-1: potential biomarkers for Parkinson’s disease, Brain 134 (Pt 7) (2011) e178. [9] R.N. Dilger, R.W. Johnson, Aging, microglial cell priming, and the discordant central inflammatory response to signals from the peripheral immune system, J. Leukoc. Biol. 84 (2008) 932–939. [10] M. Dufek, M. Hamanová, J. Lokaj, D. Goldemund, I. Rektorová, Z. Michálková, K. Sheardová, I. Rektor, Serum inflammatory biomarkers in Parkinson’s disease, Parkinsonism Relat. Disord. 15 (2009) 318–320. [11] A. Enríquez-de-Salamanca, V. Calder, J. Gao, G. Galatowicz, C. García-Vázquez, I. Fernández, M.E. Stern, Y. Diebold, M. Calonge, Cytokine responses by conjunctival epithelial cells: an in vitro model of ocular inflammation, Cytokine 44 (2008) 160–167. [12] B. Ferger, A. Leng, A. Mura, B. Hengerer, J. Feldon, Genetic ablation of tumor necrosis factor-alpha (TNF-alpha) and pharmacological inhibition of TNFsynthesis attenuates MPTP toxicity in mouse striatum, J. Neurochem. 89 (2004) 822–833. [13] K.A. Frankola, N.H. Greig, W. Luo, D. Tweedie, Targeting TNF-␣ to elucidate and ameliorate neuroinflammation in neurodegenerative diseases, CNS Neurol. Disord. Drug Targets 10 (2011) 391–403. [14] C. Gaig, E. Tolosa, When does Parkinson’s disease begin? Mov Disord. 24 (Suppl 2) (2009) S656–S664. [15] A.J. Hughes, S.E. Daniel, L. Kilford, A.J. Lees, Accuracy of clinical diagnosis of idiopathic Parkinson’s disease: a clinico-pathological study of 100 cases, J. Neurol. Neurosurg. Psychiat. 55 (1992) 181–184. [16] S. Hunot, E.C. Hirsch, Neuroinflammatory processes in Parkinson’s disease, Ann. Neurol. 53 (Suppl 3) (2003) S49–S58 (discussion S58–S60). [17] M. Irkec¸, B. Bozkurt, Epithelial cells in ocular allergy, Curr. Allergy Asthma Rep. 3 (2003) 352–357. [18] O.Y. Kwon, S.H. Kim, J.H. Kim, M.H. Kim, M.K. Ko, Schrimer test in Parkinson’s disease, J. Korean Med. Sci. 9 (1994) 239–242. [19] A.J. Lees, J. Hardy, T. Revesz, Parkinson’s disease, Lancet 373 (2009) 2055–2066. [20] M.L. Massingale, X. Li, M. Vallabhajosyula, D. Chen, Y. Wei, P.A. Asbell, Analysis of inflammatory cytokines in the tears of dry eye patients, Cornea 28 (2009) 1023–1027. [21] S.O. McGuire, Z.D. Ling, J.W. Lipton, C.E. Sortwell, T.J. Collier, P.M. Carvey, Tumor necrosis factor alpha is toxic to embryonic mesencephalic dopamine neurons, Exp. Neurol. 169 (2001) 219–230. [22] M. Menza, R.D. Dobkin, H. Marin, M.H. Mark, M. Gara, K. Bienfait, A. Dicke, A. Kusnekov, The role of inflammatory cytokines in cognition and other non-motor symptoms of Parkinson’s disease, Psychosomatics 51 (2010) 474–479. [23] M. Mogi, M. Harada, P. Riederer, H. Narabayashi, K. Fujita, T. Nagatsu, Tumor necrosis factor-alpha (TNF-alpha) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients, Neurosci. Lett. 165 (1994) 208–210. [24] M. Mogi, A. Togari, T. Kondo, Y. Mizuno, O. Komure, S. Kuno, H. Ichinose, T. Nagatsu, Caspase activities and tumor necrosis factor receptor R1 (p55) level are elevated in the substantia nigra from parkinsonian brain, J. Neural Transm. 107 (2000) 335–341. [25] S.L. Montgomery, W.J. Bowers, Tumor necrosis factor-alpha and the roles it plays in homeostatic and degenerative processes within the central nervous system, J. Neuroimmune Pharmacol. 7 (2012) 42–59. [26] D.J. Moore, A.B. West, V.L. Dawson, T.M. Dawson, Molecular pathophysiology of Parkinson’s disease, Annu. Rev. Neurosci. 28 (2005) 57–87. [27] T. Nagatsu, M. Sawada, Inflammatory process in Parkinson’s disease: role for cytokines, Curr. Pharm. Des. 11 (2005) 999–1016. [28] S. Narayanan, W.L. Miller, A.M. McDermott, Conjunctival cytokine expression in symptomatic moderate dry eye subjects, Invest. Ophthalmol. Vis. Sci. 47 (2006) 2445–2450.
Please cite this article in press as: S.S. C¸omo˘glu, et al., Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.08.019
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G Model NSL 29978 1–5
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[29] S.C. Pflugfelder, D. Jones, Z. Ji, A. Afonso, D. Monroy, Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjögren’s syndrome keratoconjunctivitis sicca, Curr. Eye Res. 19 (1999) 201–211. [30] A. Probst, A. Bloch, M. Tolnay, New insights into the pathology of Parkinson’s disease: does the peripheral autonomic system become central? Eur. J. Neurol. 15 (Suppl 1) (2008) 1–4. [31] M. Reale, C. Iarlori, A. Thomas, D. Gambi, B. Perfetti, M. Di Nicola, M. Onofrj, Peripheral cytokines profile in Parkinson’s disease, Brain Behav. Immun. 23 (2009) 55–63. [32] K. Sriram, J.M. Matheson, S.A. Benkovic, D.B. Miller, M.I. Luster, J.P. O’Callaghan, Mice deficient in TNF receptors are protected against dopaminergic neurotoxicity: implications for Parkinson’s disease, FASEB J. 16 (2002) 1474–1476. [33] C. Tamer, I.M. Melek, T. Duman, H. Oksüz, Tear film tests in Parkinson’s disease patients, Ophthalmology 112 (2005) 1795.
5
[34] M.G. Tansey, M.S. Goldberg, Neuroinflammation in Parkinson’s disease: its role in neuronal death and implications for therapeutic intervention, Neurobiol. Dis. 37 (2010) 510–518. [35] K. Varani, F. Vincenzi, A. Tosi, S. Gessi, I. Casetta, G. Granieri, P. Fazio, E. Leung, S. MacLennan, E. Granieri, P.A. Borea, A2A adenosine receptor overexpression and functionality, as well as TNF-alpha levels, correlate with motor symptoms in Parkinson’s disease, FASEB J. 24 (2010) 587–598. [36] A.D. Wahner, J.S. Sinsheimer, J.M. Bronstein, B. Ritz, Inflammatory cytokine gene polymorphisms and increased risk of Parkinson’s disease, Arch. Neurol. 64 (2007) 836–840. [37] P.S. Whitton, Inflammation as a causative factor in the aetiology of Parkinson’s disease, Br. J. Pharmacol. 150 (2007) 963–976. [38] Y.R. Wu, I.H. Feng, R.K. Lyu, K.H. Chang, Y.Y. Lin, H. Chan, F.J. Hu, G.J. Lee-Chen, C.M. Chen, Tumor necrosis factor-alpha promoter polymorphism is associated with the risk of Parkinson’s disease, Am. J. Med. Genet. B. Neuropsychiatr. Genet. 144B (2007) 300–304.
Please cite this article in press as: S.S. C¸omo˘glu, et al., Tear levels of tumor necrosis factor-alpha in patients with Parkinson’s disease, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.08.019
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