- Open Access
Long-term trihexyphenidyl exposure alters neuroimmune response and inflammation in aging rat: relevance to age and Alzheimer’s disease
- Yuqi Huang†2,
- Zhe Zhao†1,
- Xiaoli Wei1,
- Yong Zheng1,
- Jianqiang Yu2,
- Jianquan Zheng1 and
- Liyun Wang1Email author
© The Author(s). 2016
- Received: 3 February 2016
- Accepted: 22 June 2016
- Published: 1 July 2016
Clinical studies have shown an association between long-term anticholinergic (AC) drug exposure and Alzheimer’s disease (AD) pathogenesis, which has been primarily investigated in Parkinson’s disease (PD). However, long-term AC exposure as a risk factor for developing neurodegenerative disorders and the exact mechanisms and potential for disease progression remain unclear. Here, we have addressed the issue using trihexyphenidyl (THP), a commonly used AC drug in PD patients, to determine if THP can accelerate AD-like neurodegenerative progression and study potential mechanisms involved.
Male Sprague-Dawley rats (SD) were intraperitoneally injected with THP (0.3 and 1.0 mg/kg) or normal saline (NS) for 7 months. Alterations in cognitive and behavioral performance were assessed using the Morris water maze (MWM) and open field tests. After behavior tests, whole genome oligo microarrays, quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), immunohistochemistry, and immunofluorescence-confocal were used to investigate the global mechanisms underlying THP-induced neuropathology with aging.
Compared with NS controls, the MWM test results showed that THP-treated rats exhibited significantly extended mean latencies during the initial 3 months of testing; however, this behavioral deficit was restored between the fourth and sixth month of MWM testing. The same tendencies were confirmed by MWM probe and open field tests. Gene microarray analysis identified 68 (47 %) upregulated and 176 (53 %) downregulated genes in the “THP-aging” vs. “NS-aging” group. The most significant populations of genes downregulated by THP were the immune response-, antigen processing and presentation-, and major histocompatibility complex (MHC)-related genes, as validated by qRT-PCR. The decreased expression of MHC class I in THP-treated aging brains was confirmed by confocal analysis. Notably, long-term THP treatment primed hippocampal and cortical microglia to undergo an inflammatory phenotypic switch, causing microgliosis and microglia activation, which were positively accompanied by pathological misfolded tau lesions.
Our findings suggest that immune response and neuroinflammation represent a pivotal mechanism in THP-induced AD-like neuropathology processes with long-term exposure to AC drugs.
- Neuroimmune response
- Anticholinergic drug
- MHC class I
- Microglia activation
- Cognitive impairment
Negative cognitive effects are known to be an adverse effect of anticholinergic (AC) drugs and are assumed to be transient and reversible [1–3]. However, there is a new emerging hypothesis for the connection between AC exposure and pathogenesis of Alzheimer’s disease (AD), with the primary clinical lead for this connection being Parkinson’s disease (PD) [4, 5]. Trihexyphenidyl (THP) is the most commonly used AC drug in PD patients. Perry et al. found that continuous THP use for at least 2 years doubled the prevalence of both amyloid plaque and neurofibrillary tangle densities in PD patients [4–6]. Furthermore, some animal studies demonstrated that use of ACs increased Aβ peptide presence in the cortex and hippocampus, while selective M1 agonists are efficacious for AD treatment [7, 8]. Therefore, duration of AC drug administration has been described as a risk factor for appearance of dementia in PD patients. Despite early epidemiological evidence supporting this hypothesis in the clinic, understanding of the impact of ACs on pathogenesis of neurodegeneration is currently limited. In particular, it is not clear whether the onset of events that results in AC administration is part of aging or reflects early AD development. Understanding such processes is vital as most drug therapies involve chronic administration.
In the current study, we examined the hypothesis that duration of AC drug exposure affects progression of central nervous system (CNS) neurodegeneration. We treated rats with THP (0.3 or 1.0 mg/kg/day, intraperitoneal (IP)) for 7 months; these concentrations have proven successful in previous studies [9, 10]. Administration was started in 3-month-old normal Sprague-Dawley (SD) rats, while the control group was provided with normal saline (NS) only. Behavioral performance was assessed using the Morris water maze (MWM) and open field tests. Additionally, after behavioral testing, whole genome oligo microarrays and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were performed in the hippocampus to gain insight into the global gene expression and pathway responses to long-term THP exposure. Further, the cerebral cortex and hippocampus were histologically examined for associated neurodegeneration. We found that THP treatment altered neuroimmune responses and promoted CNS neuroinflammation features consistent with microgliosis and microglia activation. We believe that such detailed information is critical for understanding the long-term effects of ACs on occurrence or earlier development of AD.
Trihexyphenidyl (THP) hydrochloride was purchased from Sigma (St. Louis, MO, USA). All drugs were dissolved or diluted in NS, and solutions adjusted to a pH range of 6.5–7.0 for IP injections.
Animals and experimental design
Adult male SD rats (180–200 g) were supplied by the Animal Center of the Academy of Military Medical Science (Beijing, China). All rats were kept in plastic cages and received food and water ad libitum. The colony room was maintained at 22–23 °C and kept on a 12-h light/dark cycle. All testing was performed under the guidelines for the use of experimental animals of the Beijing Local Committee on Animal Care and Use.
Long-term THP treatment
Water maze test
A MWM apparatus (Chengdu Technology & Market Co. Ltd., Chengdu, China) was used to test spatial learning and memory, as previously described , with minor modifications. Briefly, the animals received three trials per day for 7 days to learn the task. Each trial lasted until the animal found the platform or for a maximum of 120 s. Animals that failed to find the platform within 120 s were guided there by the experimenter.
After the learning trail, rats were assigned to THP-treated (0.3 or 1.0 mg/kg; n = 10 per group) or NS-treated (n = 10) groups based on the same average escape time to reach the platform. For subsequent navigation tests, the animals underwent six consecutive training sessions for 6 months. For each training session, the animals received three trials per day for 7 days at the beginning of each month. On each trial, rats were placed into the pool facing the wall, with start locations varied across trials and days. The time taken to reach the platform was recorded as the escape latency(s). During the navigation test, escape latency to the platform for all trials was recorded. Spatial learning ability was determined by the average escape latency to the platform in every session. Swimming speed was averaged according to the last trial.
The probe trial was performed three times within 1 week following the final day of navigation training. On the day of testing, animals were given a probe trial, with the platform removed. All trials were recorded for 60 s using a digital camera. Additionally, the time spent in each target quadrant, as well as the number of annulus crossings (defined as the circular zone surrounding the platform position) was recorded to evaluate spatial memory ability. All training took place during the light phase, between 8:00 AM and 12:00 PM (lights on at 6:00 AM).
Open field activity
After completion of the MWM test, the animals were given 1 week of rest, and then spontaneous activity was assessed using a Digiscan Animal Activity Monitor system for 5 min (Jiliang Software Technology Co., Ltd., Shanghai, China), as described previously . Each activity unit contains 16-photo beams positioned 5 cm apart: 8 on the X-axis and 8 on the Y-axis. The Digiscan analyzer was interfaced with an IBM XT computer using JLBehv software (Jiliang Software Technology Co., Ltd.). On the day of testing, the rats were transferred from the animal colony into the laboratory and allowed to habituate for 120 min before testing. Total movement activity was recorded for each experiment. Inner or outer time measures the proportion of time spent in the center or outer of the arena, respectively (i.e., percentage of total time).
RNA extraction and microarray analysis
All samples were hybridized onto the Whole Rat Genome Oligo Microarray (Agilent Technologies, Madrid, Spain) encompassing more than 44,000 rat DNA probes. Protocols for the sample preparation and hybridization were adapted from the Agilent Technical Manual. RNA extraction and microarray analysis were performed as described previously . RNA was isolated using the Qiagen RNeasy Mini Kit for animal tissues (Qiagen, Inc., Valencia, CA, USA). RNA quality and quantity was checked using the Agilent 2100 bio-analyzer and by running RNA sample aliquots on the Affymetrix Gene Chip (Agilent Technologies). Total RNA was used to generate biotin-labeled cRNA using the MessageAmp™ Premier RNA Amplification Kit (cat #1972; Ambion, Austin, TX, USA). A total of 0.75 μg of biotin-labeled cRNA was hybridized onto chips by incubating at 65 °C for 17 h with constant rotation using the Eukaryotic Hybridization Control Kit (Affymetrix, San Diego, CA, USA). Arrays were washed, blocked, and labeled cRNA detected by staining with Fluidics Station 450/250 Cocktail (Affymetrix). Arrays were scanned using an Affymetrix GeneChip® Scanner 3000, and image data extracted using AGCC Software (Affymetrix GeneChip Command Console® Software). All results were given as gene expression ratio (ratio of Cy3 to Cy5 intensity). Functional annotations (including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways) were analyzed using the Molecule Annotation System (MAS) functional tool (http://bioinfo.capitalbio.com/mas3/). To detect THP-responsive genes, biological pathways that were likely to be involved in THP action in the nervous system were identified using typical criteria (fold change >2). These genes were then assigned to relevant GO biological pathways and KEGG molecular pathways by MAS. Related, significant GO biological pathways were identified by EASE score P values <0.01, while significant KEGG molecular pathways were identified using EASE score P values <0.05. Accordingly, higher enrichment and gene counts deemed that the genes of these pathways were more important.
Quantitative real-time reverse transcription-polymerase chain reaction
Sequence of specific primers used for quantitative real-time PCR (qRt-PCR)
Pair sequences (5′-3′)
A non-template control with non-genetic material was included to eliminate contamination or nonspecific reactions. Each sample (n = 4) was tested in triplicates and then used for the analysis of relative transcription data using the 2−ΔΔCT method (Table 1).
Immunohistochemistry and immunofluorescence
Paraffin wax-embedded rat brain sections (4 μm thick) were dewaxed in xylene (three times for 3 min) and rehydrated in decreasing concentrations of ethanol (100 % twice, 96 %, 70 % ethanol, and H2O twice for 3 min each), followed by washing for 5 min in 50 mM Tris-buffered saline (TBT) (pH 7.4). Optimal antigen retrieval was achieved by treating sections for 5 min with a citrate buffer (0.1 M citric acid and 0.1 M tri-sodium citrate-2-hydrate ) and microwaving at 84 °C followed by pepsin treatment, as described. Before primary antibody incubation, sections were treated for 1 h with blocking solution (phosphate-buffered saline (PBS) (pH 7.4) containing 5 % normal horse serum, 5 % normal goat serum, and 4 % BSA). Primary antibodies were diluted in PBS (pH 7.4; with 2.5 % normal horse serum, 2.5 % normal goat serum, and 2 % BSA).
Cortical or hippocampal sections from rats were incubated overnight at 4 °C in a moist chamber with rabbit CD11b (1:100), goat anti-Iba1 (1:100), rabbit p-tau AT270 (p-T181) (1:300), and phospho-tau AT8 (p-S202/T205) (1:300) in TBS-T with 1 % BSA. After a further round of washing with TBS-T, sections were incubated with DAB-conjugated secondary antibodies (1:250; Jackson ImmunoResearch, Bar Harbor, ME, USA) for 1 h in a moist chamber at room temperature. All antibodies were purchased from Abcam (Shanghai, China). Slides were then rinsed in TBS-T, mounted in a mix of glycerol/PBS (3:1), and observed using an inverted microscope (Nikon eclipse TS100, Nikon, Tokyo, Japan) equipped with a digital camera (DXM1200F).
For immunofluorescence staining, cortical or hippocampal sections were collected consecutively, and then incubated overnight at 4 °C with anti-rat MHC class I (MHCI) (ER-HR52) (1:100), mouse anti-NeuN (1:200), rabbit anti-M1 receptor (1:100), and mouse CD68 (1:100) (Abcam). After washing with PBS, sections were incubated with FITC-Green 488- or TRITC-Red 595-conjugated secondary antibody (1:500) for 2 h at 37 °C. Images were observed by laser confocal microscope (LSM710; Zeiss, Germany). Cropping of images and adjustment of brightness and contrast were identical for each staining and performed using Adobe Photoshop CS5 software (Adobe Systems, San Jose, CA, USA).
Equal protein amounts were separated on 4–12 % SDS-PAGE gradient gels, transferred to nitrocellulose membranes, and incubated overnight with primary antibody at 4 °C. The following primary antibodies were used: phospho-tau AT8 (p-S202/T205) (1:1000), p-tau AT270 (p-T181) (1:1000), CD11b (1:1000), and CD68 (1:1000). After washing, membranes were incubated with secondary antibody coupled to horseradish peroxidase. Immunocomplexes were visualized using the Super Signal West Pico Kit from GE Healthcare (Amersham). Band density measurements were obtained using ImageJ 1.36b imaging software (National Institutes of Health, Bethesda, MD, USA).
Gallyas silver staining
To identify mature neurofibrillary pathology in neurons, Gallyas silver iodide staining was performed . Sections were examined using an Olympus BX51 microscope and photographed using an Olympus camera DP-50 (Olympus, Japan).
Fluoro-Jade B staining
A commercial Fluoro-Jade B (FJB) kit (Millipore, Billerica, MA, USA) was used to detect dystrophic neuritis. Tissue sections processed for anti-NeuN immunofluorescence were mounted on gelatin-coated slides, air dried for 2–3 h, and then FJB stained in accordance with the manufacturer’s protocol. In brief, slides were incubated in a 0.06 % potassium permanganate solution for 10 min, followed by a 0.0004 % FJB solution for 30 min. Sections were washed three times in distilled water, fully dried, and cleared in xylene. Slides were coverslipped using aqueous mounting medium containing DAPI (Dako, Glostrup, Denmark) and examined under a confocal laser scanning microscope. After immersion in xylene, integrated immunofluorescence density analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The integrated pixel density was measured in three random areas from each slide (four slides in total). The average integrated pixel density was calculated for each slide and each group, and then compared.
All data were expressed as mean ± S.D. Student’s t tests were used to determine differences between the two experimental groups, while one-way or two-way analysis of variance (ANOVA) was used to compare differences between three or more groups for individual group comparisons. Data were analyzed using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA). The accepted level of significance for all tests was P < 0.05.
Cognitive behavioral impairment is present during early THP treatment.
THP affects neuroimmune/neuroinflammation genomic transcription patterns after long-term exposure
List of the top five cellular components related to different gene expression between the THP-treated and NS-treated aging rat index by the GO cellular component term
MHCI complex (MHCI)
RT1-Aw2; RT1-149; ENSRNOP00000047071; Q6MG05_RAT; RT1-CE10; ENSRNOP00000057803; Q6MG34_RAT; Q861Q3_RAT; ENSRNOP00000041900; Q6MGB8_RAT; ENSRNOP00000047152; Q6MGB9_RAT; Q6MG28_RAT; RT1-Cl; RT1-CE14; Q9JHM2_RAT; RT1-Aw2; XP_001070321; Q6MG29_RAT; Rt1.aa; XP_001072758; Q6MG32_RAT; Q861Q4_RAT; XP_001052972
Mgp; Igf2; Anxa2; Bgn; Lgals1; Igfbp2; Fmod; Lum; Ptgds; Penk; Dpp4; Oxt; Gdf10; Cartpt; Tac1; Col1a2; Fn1; DEF3B_RAT; Q6MG90_RAT; C4; Col3a1; Nid1; Rbp4; Serping1; Aebp1; ENSRNOP00000046622; Frzb; Cbln1; Kazald1; Klk8; Omd; Avp; Col1a2; Cklf; Cklf; Bmp6; Ccl6; Bmp7; Timp3; Cxcl13; Nts; Chrdl1
Lgals1; Ptgds; Lcat; Oxt; Cp; Cp; Cp; Gdf10; Cartpt; Ada; Col1a2; DEF3B_RAT; Q6MG90_RAT; C4; Rbp4; Serping1; Klk8; Lpl; Col1a2; Cklf; Cklf; Bmp6; Ccl6; Bmp7
Myh11; ENSRNOP00000047484; XP_001053321; XP_001053402; XP_001053536; XP_001073030; XP_001072354; XP_001078110; XP_001076239; XP_576790; XP_001072168; XP_576882; XP_001065390
Ptgds; Slc13a3; Dab2; Enpep; Thbd; Trpc4; ENSRNOP00000052645; ENSRNOP00000052642; ENSRNOP00000052641; ENSRNOP00000052640; ENSRNOP00000052638; ENSRNOP00000052636; ENSRNOP00000008172; ENSRNOP00000052635; Ada; Pln; RT1-Da; ENSRNOP00000003349; Colec12; XP_001061172; MGC108823; XP_574157; Thbd; Fmo1; Cklf; Cklf; ENSRNOP00000058354; INIB_RAT; RT1-Aw2; RT1-149; ENSRNOP00000047071; Q6MG05_RAT; RT1-CE10; ENSRNOP00000057803; Q6MG34_RAT; Q861Q3_RAT; ENSRNOP00000041900; Q6MGB8_RAT; ENSRNOP00000047152; Q6MGB9_RAT; Q6MG28_RAT; RT1-Cl; RT1-CE14; Q9JHM2_RAT; RT1-Aw2; XP_001070321; Q6MG29_RAT; Rt1.aa; XP_001072758; Q6MG32_RAT; Q861Q4_RAT; XP_001052972; Clic2; ENSRNOP00000001372
Tau neurodegeneration appears in the brain of rats with long-term THP exposure
Long-term THP exposure affects neuroinflammatory patterns in rats
Notably, our immunofluorescence assays revealed significant upregulation of M1 muscarinic receptor expression co-localized with CD68, which was mainly distributed on microglia/macrophages in THP-treated aging rats (Fig. 9a–c). Stereological quantification revealed the same tendency, with upregulated M1 (t = 10.37, P < 0.0001) and CD68 (t = 12.12, P < 0.0001) in the brain cortex of THP-treated aging rats compared with the age-matched NS group (Fig. 9d). These results indicate direct modulation of THP on activated microglia-linked M1 receptors.
Experimental and clinical studies consistently show that cholinergic system dysfunction has a detrimental impact on cognitive performance. Clinical studies suggest that AC agents may produce cognitive side effects among older adults, and duration of AC administration has been described as a risk factor for the appearance of dementia in PD patients. The putative mechanisms may be associated with dysregulation of cholinergic neurotransmission, thereby leading to memory impairments. Nevertheless, the exact mechanisms remain unclear. Here, we report novel findings that long-term THP administration disrupts neuroimmune signaling, with transcriptional activity of antigen processing and presentation noticeably downregulated in the hippocampus of rats. Additionally, we found exacerbated neuroinflammation with misfolded tau protein pathology related to neurodegeneration.
Aging is a multifactorial process that leads to altered behavioral function, including learning and memory. Here, we assessed AC treatment duration as a risk factor for cognitive performance, with THP administration for more than 7 months in aging rats. Thus, we assessed behavioral performance of NS-aging and THP-treated, aging rats in the MWM and open field tasks. The MWM is a task that is commonly used to assess rodents’ spatial learning and memory ability, and the normal function of the hippocampus is essential for this task [17, 18]. In the present study, animals were tested in place navigation and probe trials. It should be noted that only rats treated with THP (1.0 mg/kg, IP) displayed a significantly poor performance in spatial tasks during the initial 3-month training session, in which escape latency was significantly disrupted. It is known that THP acts as a muscarinic receptor antagonist, and muscarinic acetylcholine receptors play key roles in facilitating cognitive processes. This impairment is consistent with several studies using nonselective muscarinic receptor antagonists to block cholinergic signaling in aging and AD rodent models [19, 20]. However, we did not find memory disruption among THP-dosed and NS age-matched groups upon repetition of MWM training following subsequent treatments, with escape latency or swimming speed showing no significant impairment. Moreover, after the final place navigation training, all tested groups generated similar path length percentages during the probe task, while the THP (0.5 mg/kg) group spent significantly more time in the target quadrant of the apparatus compared with the other two groups. The probe data confirm that memory disruption is only present early on in THP treatment. In addition, after the MWM test, THP- and NS-treated rats showed no significant behavioral discrepancies in the open field test. THP and the behavioral deficit in memory retrieval at the start of place navigation may reflect its acute function in blocking muscarinic receptors.
Using microarray analysis, the global impact of THP on the hippocampal gene expression was examined. GO and KEGG pathway analysis showed that the most significantly affected GO category for long-term THP treatment was immune response and antigen processing and presentation or neuroimmune/neuroinflammatory functions. In particular, genes related to the function of the MHC class I protein complex were significantly downregulated (Table 2 and Fig. 4b). These data were verified by qRT-PCR and confocal-immunofluorescence analyses, with MHC-related genes and MHCI expression significantly downregulated in the rat brain (Fig. 5a, b). Hence, our microarray findings show that long-term THP treatment suppresses innate and adaptive immune homeostasis in laboratory animals. This indicates that THP may function either directly or indirectly in several immune responses and neuroinflammation.
Next, we found that subchronic THP exposure could prime pronounced neuroimmune/neuroinflammatory dysfunction and neuronal phenotypic changes. Neuroinflammatory abnormalities represent a key difference between THP- and NS-treated, age-matched rats. Specifically, THP-treated rats display significantly more Iba1- and CD11b-positive cells of phagocytic morphology compared with age-matched NS-treated littermates. Immunoblotting confirmed the increase in CD11b- and CD68-positive cells associated with long-term systemic THP challenge.
Notably, our current study shows that in activated microglia, THP specifically upregulates M1 muscarinic receptor expression, with a concomitant increase in CD68 (Fig. 9c). THP is widely used as a nonselective muscarinic antagonist and inhibits acetylcholine transmission via blockage of mAChRs [7, 8]. Our current findings suggest that long-term THP use may upregulate muscarinic M1 receptor expression on microglia, and thereby play a role in stimulating phenotypic changes in microglia. Although we cannot exclude the involvement of other muscarinic receptor types in the observed THP effects on microglia, our results support our previous links between cholinergic modulation and the immune system and inflammation.
Involvement of inflammation is considered to be an important factor in resistance variability or susceptibility to AD [21, 22]. Here, we found different alterations in tau pathology spreading in an AD-like manner. In normal young rat brain (3 months old), there was less tau hyperphosphorylation and neurofibrillary tangles. Notably, rats with subchronic THP exposure showed vulnerability to neurofibrillary degeneration with misfolded tau protein aggregation compared with NS-treated, age-matched rats. Stereological quantification and immunoblotting revealed higher levels of phosphorylated tau AT8 and AT270 changes in THP-treated rats compared with age-matched controls. Long-term THP also accelerated a mature neurofibrillary pathology and neurodegeneration in neurons, and further, confirmed by Gallyas silver and FJB staining in the hippocampus of rat brain. Our study supports the hypothesis that long-term THP exposure affects the progression of neurodegeneration in these aging animals.
Despite the evidence described above, neurofibrillary degeneration coupled with an inflammatory response is not consistent with the cognitive behavioral performance. Our study shows that cognitive performance is disrupted as an acute impairment and is only present early on in THP treatment, likely as a consequence of cholinergic signaling blockage because of AC activity. Notably, this behavioral deficit did not continue or exacerbate upon repeated behavioral tests with subsequent THP treatment. Indeed, performances in the MWM and open field tests suggest that chronic THP exposure could lead to adaptation alteration or may be a compensatory mechanism to support cognitive performance, even with loss of neuronal activity. Accordingly, our study supports the hypothesis that THP impacts on the pathogenesis of neurodegeneration in laboratory animals. However, the neuropathological alterations related to these adaptations have yet to be confirmed. Additionally, our current study indicated THP-induced neuroimmune/neuroinflammatory dysfunction may be an event leading to earlier AD development. The neurodegenerative neurofibrillary pathogenesis that results from THP administration might be an earlier event according to the performance in behavioral tests. Moreover, there has been no systematic confirmation that acute or chronic prescription of such medications leads to transient or permanent adverse cognitive outcomes. Several gaps remain in the existing literature on the previously described association between AC use and clinical cognitive function. The mechanisms of action of medications determined in previous studies are inconsistent, making generation of a comprehensive, clinically useful list of AC medications impractical based on those data sets [23, 24]. Thus, understanding such processes is vital because most drug therapies involve chronic AC administration. Our current study is the first to comprehensively measure the cognitive impact of long-term AC exposure on AD in aged laboratory animals. Neuroimmune/neuroinflammatory dysfunction is presented as one of the key differences between THP- and age-matched, NS-treated aging rats, accompanying deleterious neurodegenerative progression. AD is the most common form of dementia in the older population and is characterized by progressive neurodegeneration of the CNS. While the precise etiology of this disease still remains unknown, it is believed that intracellular accumulation of hyperphosphorylated tau (which forms neurofibrillary tangles) and deposition of extracellular filaments play a critical role in neurodegeneration. Accordingly, tau pathologies are evident in AD [21, 25, 26]. From a molecular perspective, AD is a multifactorial disorder involving the association of genetic and environmental factors. Onset and progression of AD may be influenced by several risk factors such as hypertension, metabolic disorders, and/or inflammatory status [27–29]. Owing to the limitations of our study, we cannot clarify the nature of interactions among THP, CNS inflammatory compartments, and neuronal neurodegeneration in this model. Future studies are necessary to determine how THP induces switching of systemic inflammatory events and progression of neurodegenerative pathology. Furthermore, additional studies examining the mechanism by which THP exerts its effects in more commonly used AD models should be performed. Nonetheless, our current results, together with the literature, provide clear implications for the rational use of ACs in older patients.
In summary, to our knowledge, this is the first study to clearly show that long-term THP treatment impacts on genes related to immune response pathways, particularly antigen presentation, and propagates neuroimmune/neuroinflammatory dysfunction. Our findings indicate that a potential mechanism of THP relates to its effects on several aspects of the immune response, either directly or via neuroimmune pathways. These effects may play a role in modulating the progression of AD and related neurodegenerative disorders.
AC, anticholinergic; AD, Alzheimer’s disease; CNS, central nervous system; FJB, Fluoro-Jade B; Iba1, ionized calcium binding adaptor molecule 1; IHC, immunohistochemical; IP, intraperitoneal; MHC, major histocompatibility complex; MWM, Morris water maze; NS, normal saline; PBS, phosphate-buffered saline; PD, Parkinson’s disease; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; SD, Sprague-Dawley rats; TBT, Tris-buffered saline; THP, trihexyphenidyl
This work was supported by the National Mega-Project of Science Research of China (2009ZX09102-007), the National Integrated Drug Discovery Technology Platform Foundation of China (2012ZX09301003), and the Open project of Key Laboratory for Neurodegenerative Diseases of Ministry of Education (2013SJBX03). All sources of funding for the research declare that they have no competing financial or personal interests and that none of the author’s institutions have contracts relating to this research through which it may stand to gain financially now or in the future.
Availability of data and materials
The datasets and materials supporting the conclusions of this article are included within the article and its additional files.
YH and ZZ contributed equally to the work. LW participated in the research design and performed the data analysis. LW and JY wrote or contributed to the writing of the manuscript. YH, ZZ, XW, and ZY conducted the experiments. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Consent for publication is not applicable for this manuscript. Our manuscript does not contain any individual person’s data in any form (including individual details and images); we do not need any consent form from individual person or legal guardian for publication.
Ethics approval and consent to participate
All animal experimentation was approved by the Beijing Local Committee on Animal Care and Use. All experiments were performed according to the Guide for the Care and Use of Experimental Animals of the Beijing Local Committee and the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1985).
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- Flicker C, Serby M, Ferris SH. Scopolamine effects on memory, language, visuospatial praxis and psychomotor speed. Psychopharmacology (Berl). 1990;100:243–50.View ArticleGoogle Scholar
- Ancelin ML, Artero S, Portet F, Dupuy AM, Touchon J, Rithie K. Nondegenerative mild cognitive impairment in elderly people and use of anticholinergic drugs: longitudinal cohort study. BMJ. 2006;332:455–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Boustani M, Campbell NL, Munger S, Maidment I, Fox C. Impact of anticholinergics on the aging brain: a review and practical application. Aging Health. 2008;4:311–20.View ArticleGoogle Scholar
- Perry EK, Kilford L, Lees AJ, Burn DJ, Perry RH. Increased Alzheimer pathology in Parkinson’s disease related to antimuscarinic drugs. Ann Neurol. 2003;54:235–8.View ArticlePubMedGoogle Scholar
- Kajimura N, Mizuki Y, Kai S, Suetsugi M, Yamada M, Okuma T. Memory and cognitive impairments in a case of long-term trihexyphenidyl abuse. Pharmacopsychiatry. 1993;26:59–62.View ArticlePubMedGoogle Scholar
- Takahashi S, Tohgi H, Yonezawa H, Obara S, Yamazaki E. The effect of trihexyphenidyl, an anticholinergic agent, on regional cerebral blood flow and oxygen metabolism in patients with Parkinson’s disease. J Neurol Sci. 1999;167:56–61.View ArticlePubMedGoogle Scholar
- Welt T, Kulic L, Hoey SE, McAfoose J, Späni C, Chadha AS, Fisher A, Nitsch RM. Acute effects of muscarinic M1 receptor modulation on AβPP metabolism and amyloid-β levels in vivo: a microdialysis study. J Alzheimers Dis. 2015;46:971–82.View ArticlePubMedGoogle Scholar
- Haring R, Fisher A, Marciano D, Pittel Z, Kloog Y, Zuckerman A, et al. Mitogen-activated protein kinase-dependent and protein kinase C-dependent pathways link the m1 muscarinic receptor to b-amyloid protein secretion. J Neurochem. 1998;71:2094–103.View ArticlePubMedGoogle Scholar
- Tanda G, Ebbs AL, Kopajtic TA, Elias LM, Campbell BL, Newman AH, Katz JL. Effects of muscarinic M1 receptor blockade on cocaine-induced elevations of brain dopamine levels and locomotor behavior in rats. J Pharmacol Exp Ther. 2007;321:334–44.View ArticlePubMedGoogle Scholar
- Bédard MA, Pillon B, Dubois B, Duchesne N, Masson H, Agid Y. Acute and long-term administration of anticholinergics in Parkinson's disease: specific effects on the subcortico-frontal syndrome. Brain Cogn. 1999;40:289–313.View ArticlePubMedGoogle Scholar
- Nelson RL, Guo Z, Halagappa VM, Pearson M, Gray AJ, et al. Prophylactic treatment with paroxetine ameliorates behavioral deficits and retards the development of amyloid and tau pathologies in 36TgAD mice. Exp Neurol. 2007;205:166–76.View ArticlePubMedPubMed CentralGoogle Scholar
- Boger HA, Middaugh LD, Huang P, Zaman V, Smith AC, Hoffer BJ, Tomac AC, Granholm AC. A partial GDNF depletion leads to earlier age-related deterioration of motor function and tyrosine hydroxylase expression in the substantia nigra. Exp Neurol. 2006;202:336–47.View ArticlePubMedGoogle Scholar
- Martin B, Pearson M, Brenneman R, Golden E, Wood W, et al. Gonadal transcriptome alterations in response to dietary energy intake: sensing the reproductive environment. PLoS One. 2009. doi:10.1371/journal.pone.0004146.Google Scholar
- Ding L et al. Human four-and-a-half LIM family members suppress tumor cell growth through a TGF-β-like signaling pathway. J Clin Invest. 2009;119:349–61.PubMedPubMed CentralGoogle Scholar
- Krstic D, Madhusudan A, Doehner J, Vogel P, Notter T, Imhof C, Manalastas A, Hilfiker M, Pfister S, Schwerdel C, Riether C, Meyer U, Knuesel I. Systemic immune challenges trigger and drive Alzheimer-like neuropathology in mice. J Neuroinflammation. 2012;2(I):9–151.Google Scholar
- Gallyas F. Silver staining of Alzheimer’s neurofibrillary changes by means of physical development. Acta Morphol Acad Sci Hung. 1971;19:1–8.PubMedGoogle Scholar
- Moser MB, Moser EI. Distributed encoding and retrieval of spatial memory in the hippocampus. J Neurosci. 1998;18:7535–42.PubMedGoogle Scholar
- Terry Jr AV. Spatial navigation (water maze) tasks. In: Buccafusco JJ, editor. Methods of behavior analysis in neuroscience. Boca Raton (FL): CRC Press/Taylor & Francis; 2009. Chapter 13.Google Scholar
- Klinkenberg I, Blokland A. The validity of scopolamine as a pharmacological model for cognitive impairment: a review of animal behavioral studies. Neurosci Biobehav Rev. 2010;34:1307–50.View ArticlePubMedGoogle Scholar
- Blokland A. Acetylcholine: a neurotransmitter for learning and memory? Brain Res Rev. 1995;21:285–300.View ArticlePubMedGoogle Scholar
- Stozicka Z, Zilka N, Novak P, Kovacech B, Bugos O, Novak M. Genetic background modifies neurodegeneration and neuroinflammation driven by misfolded human tau protein in rat model of tauopathy: implication for immunomodulatory approach to Alzheimer’s disease. J Neuroinflammation. 2010. doi:10.1186/1742-2094-7-64.PubMedPubMed CentralGoogle Scholar
- Perry VH, Teeling J. Microglia and macrophages of the central nervous system: the contribution of microglia priming and systemic inflammation to chronic neurodegeneration. Semin Immunopathol. 2013;35:601–12.View ArticlePubMedPubMed CentralGoogle Scholar
- Fox C, Livingston G, Maidment ID, Coulton S, Smithard DG, Boustani M, Katona C. The impact of anticholinergic burden in Alzheimer's dementia-the LASER-AD study. Age Ageing. 2011;40:730–5.Google Scholar
- Cai X, Campbell N, Khan B, Callahan C, Boustani M. Long-term anticholinergic use and the aging brain. Alzheimers Dement. 2013;9:377–85.View ArticlePubMedGoogle Scholar
- Simón D, Hernández F, Avila J. The involvement of cholinergic neurons in the spreading of tau pathology. Front Neurol. 2013. doi:10.3389/fneur.2013.00074.PubMedPubMed CentralGoogle Scholar
- Zilka N, Kazmerova Z, Jadhav S, Neradil P, Madari A, Obetkova D, Bugos O, Novak M. Who fans the flames of Alzheimer’s disease brains? Misfolded tau on the crossroad of neurodegenerative and inflammatory pathways. J Neuroinflammation. 2012;9:47.View ArticlePubMedPubMed CentralGoogle Scholar
- Casserly I, Topol E. Convergence of atherosclerosis and Alzheimer’s disease: inflammation, cholesterol, and misfolded proteins. Lancet. 2004;363:1139–46.View ArticlePubMedGoogle Scholar
- Ryman D, Lamb BT. Genetic and environmental modifiers of Alzheimer’s disease phenotypes in the mouse. Curr Alzheimer Res. 2006;3:465–73.View ArticlePubMedGoogle Scholar
- Stozicka Z, Zilka N, Novak M. Risk and protective factors for sporadic Alzheimer’s disease. Acta Virol. 2007;51:205–22.PubMedGoogle Scholar