The role of microglia in thalamic reticular nucleus in acupuncture regulating cognitive deficits in insomnia rats

Wei Ting (韦婷), Li Ming (黎明), Bai Ai-ling (白艾灵), Liu Yi (刘祎), Zhao Zheng-yu (赵征宇), Cai Ding-jun (蔡定均) Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Sichuan 610075, China

Abstract

Keywords: Acupuncture Therapy; Microglia; Cognitive Dysfunction; Thalamic Reticular Nucleus; Event-related Potentials, P300; Insomnia; Rats

Sleep is critical to human survival and development. As a physiological process of life, effective sleep is necessary to maintain high cognitive performance during waking. Insomnia may affect people of all ages and is a common disease that endangers health, and about 27% of people in the world suffer from insomnia[1]. Clinical epidemiological studies have suggested that insomnia is a risk factor for cardiovascular disease[2], mental disorder, including anxiety and depression[3], as well as inflammatory disease[4]. Sleep disorders are closely related to cognitive deficits such as concentration disturbances, memory loss, and decreased alertness[5]. Insomniacs with an objective sleep time below 6 h present significant cognitive deficits, mainly manifested as the decline of attention, situational memory, executive function and social ability[6]. What is more serious is that cognitive deficits caused by insomnia are the main cause of general accidental injuries and fatal injuries due to motor vehicles[7]. Cognitive mechanisms are instantiated by the dynamic interplay of brain regions forming task-dependent networks. Attention is a cognitive function, as the precursor of cognitive process, which includes the recessive change of nerve representation of sensory stimuli without obvious behavioral changes[8]. Thalamic reticular nucleus (TRN) is the main inhibitor of thalamic relay nucleus and is essential for the thalamocortical pathway. Accumulated evidences have indicated that TRN not only regulates the sleep-wake cycle[9], but also plays an important role in attention regulation[10].

As a major component of traditional Chinese medicine, acupuncture is an effective way to treat insomnia. The systematic reviews indicated that acupuncture could be effective against insomnia[11-12]. A randomized controlled trial (RCT) implied that acupuncture treatment was more effective than sham acupuncture treatment in increasing insomnia patients' sleep quality and improving their psychological health[13]. Acupuncture could significantly improve sleep efficiency and total sleep time, and was superior to oral zopiclone tablets[14]. Our previous clinical research found that acupuncture could adjust the macroscopic structure of sleep in patients with primary insomnia, and regulate the sleep-wake cycle, as well as integrate the disordered sleep microstructure to enhance sleep stability[15]. Meanwhile, acupuncture has exhibited a potent effect on regulating the concomitant symptoms associated with insomnia including cognitive deficits. The P300 (or P3), as an important member of event- related potentials (ERPs), has been widely used in cognitive function testing on attention, memory and other aspects[16-17]. Previous studies have found that patients with primary insomnia have prolonged P3 latency, decreased P3 peak amplitude, and increased the Pittsburgh sleep quality index (PSQI) global score. Acupuncture can significantly shorten the P3 latency and increase P3 amplitude in insomnia patients, and lead to an obvious decrease in the PSQI global score. The results showed that acupuncture could not only increase the quality of sleep in patients with insomnia, but also improve the cognitive function in patients with insomnia such as memory loss and attention deficit[18-19]. Acupuncture has a benign regulatory effect on cognitive deficits after insomnia, while the mechanism has not been fully elucidated. Therefore, we focused on microglia in the TRN region to explore the possible mechanism about acupuncture treatment in treating cognitive deficits due to insomnia in this study.

1 Materials and Methods

1.1 Experimental animals and grouping

Male Sprague-Dawley rats, at 7-8 weeks old, weighing 200-240 g, were provided by Chengdu Dashuo Experimental Animal Co., Ltd., China [License No.: SCXK (Chuan) 2015-030]. The rats were domesticated in the isolation unit of the Acupuncture & Chronobiology Laboratory of Chengdu University of Traditional Chinese Medicine, with free access to food and water. Automatic light and dark control, 7:00-19:00 for the light period, room temperature (22±1) ℃, relative humidity 50%-60%, automatically ventilated once every 1 h. After 1 week of domestication, 30 rats having adapted to light-dark cycles were selected. According to the average activity during the light period, they were randomly divided into 10 rats in the control group and 20 rats for model establishment. After the models were successfully established, the model rats were completely randomly divided into a model group and an acupuncture group, with 10 rats in each group. The various treatments of animals during the experiment were in line with theGuiding Opinions on the Treatment of Experimental Animalspromulgated by the Ministry of Science and Technology of the People's Republic of China in 2006.

1.2 Main reagents and instruments

CLOCKLAB 2 biological rhythm data acquisition and analysis system (Coulbourn, USA); Brain Amp® amplifier, ERPs detection instrument Brain Vision Recorder 2 and Brain Vision Analyzer 2 (Brain Products, Germany); electrode needle and junction box (Hanxiang Biomedical Electronics Co., Ltd., China); fluorescence scanning microscope (Pannoramic 250 Flash Ⅱ, 3D Histech, Hungary); microplate reader (Meigu Molecular Instruments Co., Ltd., China); para-chlorophenylalanine (PCPA, Batch No.: SHBJ4632, Sigma, USA); rat interleukin (IL)-1β enzyme-linked immunesorbent assay (ELISA) kit (Cat. No.: ZC-36391) and rat tumor necrosis factor (TNF)-α ELISA kit (Cat. No.: ZC-37624, Zhuocai Biotechnology Co., Ltd., China); mouse monoclonal antibody ionised calcium binding adaptor molecule 1 (Iba-1, Batch No.: GB12105, Servicebio, China); rhodamine labeled goat anti-mouse IgG (Batch No.: ZF-0313, Beijing Zhongshang Jinqiao Biological Co., Ltd., China); chloral hydrate (Batch No.: Q12HB 4218-2009, Comemi Chemical Reagent Co., Ltd., China).

1.3 Model development

Rats were injected with PCPA suspension [300 mg/(kg·bw)] at zeitgeber time 4 (ZT4, 11:00 a.m., lights on at 7 a.m.) to establish insomnia model once a day for 2 d[20]. Rats in the control group were intraperitoneally injected with the same amount of normal saline at ZT4. The insomnia rats models were concluded to be successful or not through the spontaneous activity of rats during the light period.

1.4 Grouping and interventions

Acupuncture treatment began on the first day after PCPA injection and lasted for 5 d. According to the results of the previous study, the rats were treated with acupuncture at ZT4. They were put into fixation- machine for 15 min, and needles were inserted at Neiguan (PC 6) to a depth of 1 mm and Zusanli (ST 36) to a depth of 7 mm. Referring theExperimental Acupuncture Science[21], Neiguan (PC 6) is located on the inside of the forelimb, between the ulna and the tibia about 3 mm away from the wrist joint, and Zusanli (ST 36) is located on the posterior aspect of the knee joint, 5 mm beneath the capitulum fibulae. The control group and the model group were synchronously bundled and fixed for 15 min at ZT4.

1.5 Observation indicators and detection methods

1.5.1 Analysis methods of spontaneous activity data

The CLOCKLAB 2 data acquisition system was used to dynamically monitor the 24-hour spontaneous circadian rhythm of rats, and the analysis system was used to analyze the spontaneous treadmill activity data of rats. According to the changes in circadian rhythm parameters of spontaneous activity in the rats, the sleep quality of rats was evaluated. Then, the changes in spontaneous activity during the light period were used to judge whether the rats suffered from insomnia. 1.5.2 Record and analysis of electroencephalograph (EEG)

EEG was recorded using the event-related potential detectors Brain Vision Recorder 2 and Brain Amp® amplifiers. The EEG recording process was performed in a bioelectrical laboratory that was insulative and homothermal. Rats in each group were anesthetized by intraperitoneal injection of 10% chloral hydrate [3.5 mL/(kg·bw)]. The stimulus presentation was

All data were presented as mean ± standard deviation (±s). Statistical analyses were performed controlled by the E-prime 2.0 experimental software. The recording electrode needle was inserted under the scalp of the intersection of the forehead line and the two external auditory canal lines. The reference electrode needle was placed at the tip of the nose, and the ground electrode needle was inserted into the tail of the rat. The earphones were fixed at 2 cm from the external auditory canal. Make sure that the resistance of the electrode needle and the local contact part falls below 5 kΩ. The sampling rate of the EEG signal was 1 000 Hz. The filter bandpass was 0.1-100 Hz and the resolution was 0.1 μV. P3 was induced by auditory oddball mode. The probability of non-target stimulation S1 (frequency 1 000 Hz, 60 dB, 120 ms) was 80% and the probability of target stimulation S2 (frequency 2 000 Hz, 95 dB, 120 ms) was 20%. Twenty sequences were recorded per rat. Brain Vision Analyzer 2 was used to analyze the EEG data. The P3 time window was defined as the maximum positive peak within 250-500 ms.

1.5.3 Immunofluorescence

After treatment on the 5th day, the rats were anaesthetized as previously described and perfused via the aorta with 200 mL normal saline, followed by 250 mL 4% paraformaldehyde in 0.1 mol/L phosphate- buffered saline (PBS). The whole brain was extracted on an ice platform and placed in 4% paraformaldehyde fixative. Paraffin sections were mounted on poly-L-lysine-coated slides for immunofluorescence. They were deparaffinized with xylene. After washing, the sections were blocked with 3% H2O2for 10 min and treated with a sodium citrate buffer at 95 ℃ for antigen retrieval for 20 min. After being blocked in 10% newborn bovine serum for 1 h at 37 ℃, microglia was detected using mouse monoclonal antibody ionised calcium binding Iba-1 (a microglia marker) at 4 ℃ overnight. Then the sections were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody for 1 h at 37 ℃. Chromosomes were labeled with 10 g/mL DAPI and observed under a fluorescence scanning microscope.

1.5.4 Assessment of cytokines

After 5-day intervention, rats were anesthetized by 10% chloral hydrate [350 mg/(kg·bw)]. The brain tissue was quickly taken out on the ice table and rinsed with normal saline. The TRN area was separated and stored in a liquid nitrogen tank. And then the tissue samples were immediately immersed in liquid nitrogen. The IL-1β and TNF-α were determined in a 48-well micro- plate using a sandwich ELISA kit. The absorbance was measured at 450 nm on a microplate reader and the concentrations of IL-1β and TNF-α were calculated.

1.6 Statistical methods

using SPSS 21.0 software.Comparisonsbetween groups were performed using one-way analysis of variance with Student-Newman-Keuls correction for multiple comparisons.P＜0.05 was considered to indicate a statistically significant difference.

2 Results

2.1 The effect of acupuncture on spontaneous activity during the light period of rats

During domestication period, the three groups of rats were resting during the light period and active during the dark period, with spontaneous activities presenting obvious circadian rhythm.There was no significant difference in the amount of spontaneousactivity during the light period acrossthe groupsduring this period(allP＞0.05).After modeling,the spontaneous circadian rhythm in the model group and the acupuncture group disappeared,and the spontaneous activity during the light period increased significantly (bothP＜0.01).After treatment,the spontaneous circadian rhythm of rats in the acupuncture group gradually recovered,and the rats in the acupuncture group had less spontaneous activity during the light period than those in the model group(P＜0.01).The results strongly showed that acupuncture could effectively improve the sleep status in insomnia rats.Please check Figure 1 and Figure 2.

Figure 1.Histogram of circadian rhythm of spontaneous activity of rats in each group

Figure 2.Circadian rhythm of spontaneous activity of rats

2.2 Effect of acupuncture on cognitive function of rats

The P3 is known to be affected virtually in all neurological disorders, and the prolongation of P3 latency and the decrease of amplitude are the main manifestations of EEG detection in patients with cognitive impairment such as attention deficit and memory loss[16,22].At the baseline,there was no significant difference in the P3 latency and amplitude among the three groups before the model establishment (allP＞0.05).After modeling,the P3 latency was significantly longer (bothP＜0.05), and the P3 amplitude decreased(bothP＜0.01).After acupuncture treatment, the latency and amplitude of P3 presented significant changes in the acupuncture group compared with the model group (bothP＜0.05).P3 latency and amplitude showed no significant changes in the control group (bothP＞0.05). These data evidenced that acupuncture could improve cognitive deficit by regulating the P3 latency and amplitude. The details are shown in Figure 3, Figure 4 and Figure 5.

Figure 3. Changes in the ERPs of rats in each group

Figure 5.Comparison of P3 amplitude of rats in each group

2.3 Effect of acupuncture on microglia activation in TRNof insomnia rats

The activation of microglia in the TRN region was assessed via immunofluorescence.In the control group,the number of Iba-1 positive cells was smaller and presented lower optical density at the resting stage in the TRNregion.Whereas,the Iba-1 positive cells in the model group were remarkably more at the activated stage,with the cell branches becoming shorter and thicker,and the average optical density value increased significantly (P＜0.01).After acupuncture treatment,we observed remarkable improvement in microglia cell morphology,and the average optical density value in the acupuncture group wassignificantly lower than that in the model group(P＜0.05). These results suggested that insomnia stimulated microglia activation,while acupuncture treatment inhibited the activated stage of microglia.Please check Figure 6 and Figure 7.

2.4 Effect of acupuncture on inflammatory factors IL-1βand TNF-α in the TRN region of insomnia rats

In humans and rodents,sleep loss increases the levels of pro-inflammatory mediators such as IL-1βand TNF-α[23],and inflammation is a key factor leading to cognitive impairment.The levels of inflammatory cytokines IL-1βand TNF-α were detected by ELISA in this study.Compared with the control group,the concentrations of IL-1β and TNF-α in the model group increased significantly (bothP＜0.05).The concentrations of IL-1β and TNF-α in the acupuncture group were remarkably lower than those in the model group(bothP＜0.05).There were no significant differences in the concentrations of IL-1β and TNF-α between the acupuncture group and the control group(bothP＞0.05).The results showed that insomnia increased the concentrationsof IL-1βand TNF-α in TRN,while acupuncture could significantly reduce the levels of these inflammatory factors (Figure 8 and Figure 9).

3 Discussion

The activity of the GABAergic neurons of TRN has long been known to play crucial roles in modulating the transmission of information through the thalamus and in generating changes in thalamic activity during transitions from wakefulness to sleep[24].TRN,as a gate connecting thalamus and cortex,plays an irreplaceable role in sleep and attention regulation because of its special nerve structure and important anatomical location.The cerebralcortex and the thalamus maintain a dynamic balance between various body functions,including sleep,wakefulness and attention,through different direct or indirect connections.In addition,a number of studies have shown that round-trip axons between thalamus and cortex almost entirely passing through the TRN,which is thus regarded as the gate for screening various sensory information between thalamus and cortex[25-26].Some studies have shown that ‘leaky thalamus’caused by impaired TRN function may be the basis of attention deficit and sleep interruption.There is a great overlap between the TRN circuit involved in sleep-regulating spindle waves and the TRN circuit that attracts attention.Therefore,it is speculated that insomnia and attention deficits may be caused by common TRNcircuit dysfunction[27-28].

Figure 6.Comparison of microglia activation in the TRN region of rats in each group

Figure 7.Comparison of microglia activation in the TRN region of rats(immunofluorescence,×400)

Figure 8.Comparison of the IL-1βconcentration in the TRN region of rats in each group

Figure9.Comparison of the TNF-α concentration in the TRN region of ratsin each group

Microglia is a crucial regulator of brain development and homeostasis via neuronal-microglial interactions,synaptic modeling,scavenging of cellular debris and secretion of tropic factors.Once the central nervous system isexposed to environmentalrisk factors(such as infection,chronic psychological stress,air pollution,etc.),it has profound effects on microglia and the inflammatory milieu in the brain,as well as cognition[29-30].Previous study has demonstrated that impaired sleep affects adaptive immune responses and interferes with the steady state of the brain,leading to microglial activation and elevated pro-inflammatory mediators[31].Increasing evidences have demonstrated that sleep deprivation induces the activation of microglia in brain region including hypothalamus,hippocampus and cortex,which results in a great release of pro-inflammatory mediators including TNF-α and IL-1β.In turn,the pro- inflammatory mediators maintain microglial activation,which results in a vicious circle,thereby impairing cognitive function[32-34].However,after treatment with minocycline during sleep deprivation,microglial activation is inhibited and inflammatory responses are reduced,which will improve the neurogenesis and cognitive performance[34].Our study also found that the levels of inflammatory mediators TNF-α and IL-1βin the TRN region of insomnia rats were significantly increased,microglia was significantly activated,and the insomnia rats showed prolonged latency and decreased amplitude in P3.These results suggested that insomnia stimulated microglial activation,resulting in proinflammatory mediators release and induced cognitive impairment.

Increasing evidence has confirmed that acupuncture can inhibit the activation of microglia in dementia rats and reduce the expression levels of central TNF-α,IL-1β and other inflammatory factors,which will increase spatial learning and memory in the rats and improve cognitive performance[35-36].Our previous study supported that acupuncture possessed an ability to correct the attention-related cognitive deficits in patients with insomnia[18].Moreover, in this study,we showed the inhibitory effect of acupuncture on microglial activation and the release of pro-inflammatory mediators in TRN due to insomnia.Importantly,acupuncture could remarkably improve the latency and amplitude of P3,as well as the sleep rhythm.These findings demonstrated that acupuncture could effectively inhibit the activation of microglia in the TRNregion,and reduce the inflammatory level, thereby improving the cognitive deficit due to insomnia in rats.

Traditional Chinese medicine believes that‘inharmonious stomach leads to insomnia’.The combination of Neiguan(PC 6)and Zusanli (ST 36)has the functionsof regulating qi,harmonizing stomach and tranquilizing mind.They are the frequently-used acupoints for treating insomnia and accompanied symptoms after insomnia.Our results indicated that acupuncture at the two points improved the sleep quality and cognitive function in insomnia rats.Some studies have reported that stimulating Neiguan(PC 6)can change the amplitude of the intrinsic cortical activity of the brain and increase connections between cerebral cortex regions.Thus,stimulating Neiguan (PC 6)may be an effective way for improving cognition[37-38].

In conclusion,the study presented strong evidence that acupuncture had an effect on improving the cognitive deficit of insomnia rats by inhibiting microglial activation and reducing inflammatory mediators in the TRN region.Therefore,acupuncture at Neiguan(PC 6)and Zusanli(ST 36)can be considered as an effective way to treat cognitive deficit due to insomnia.

Conflict of Interest

There isno potential conflict of interest in thisarticle.

Acknowledgments

This work was supported by National Natural Science Foundation of China (国家自然科学基金项目,No.81373559, No.81774434).

Statement of Human and Animal Rights

The treatment of animals conformed to the ethical criteria.All procedures and animal experiment were approved by the Animal Care and Use Committee of the Chengdu University of Traditional Chinese Medicine(Chengdu,China, No.2014-01).Received:22 January 2020/Accepted:5 March 2020