The protective effect of PK11195 on D-galactose-induced amnestic mild cognitive impairment in rats

Introduction

Early cognitive decline is increasing in prevalence with the growth of the aging population in China. Premature changes in cognitive function differ from those associated with normal or accelerated aging; although multi-dimensional cognitive decline occurs in these individuals with age, under normal circumstances, the damage to cognitive dimensions is more balanced. Mild cognitive impairment (MCI) is defined as a state in which the level of cognition declines beyond that of normal aging, but dysfunction has not yet occurred (1). Amnestic mild cognitive impairment (aMCI) is associated with frequent memory loss and progression to Alzheimer’s disease (AD) (2). Epidemiological studies have reported that approximately half of patients who experience a mild decline in learning and memory abilities go on to develop AD (3). In 2018, healthcare, long-term care, and hospice services for AD patients aged ≥65 years in the United States totaled approximately $277 billion (4). Therefore, exploring ways to prevent, diagnose, and treat aMCI is of immense value to reducing the occurrence of AD.

aMCI is a normal part of the brain’s natural aging process. Simulated aging has been widely used in in vivo studies; in AD mechanism research and drug model screening, chronic exposure to D-galactose (D-gal) has often been used to induce neurotoxicity in rat models (5,6). With natural aging, the synthetic density of translocator protein 18kDa (TSPO) in the brains of animals continues to increase (7). TSPO is a common mitochondrial protein of 18kDa in size. It was originally named peripheral-type benzodiazepine receptor (PBR) and is widely distributed in many organs and tissues including the brain, kidney, and heart (8). TSPO is mainly located on the mitochondrial membrane of external steroid synthesis cells. Among its main functions is the transportation of cholesterol into the mitochondria, which is a prerequisite for mitochondrial steroid synthesis (9). In a number of neuropathological studies, TSPO was used as a biomarker of neuroinflammation and reactive glial hyperplasia; consequently, various TSPO ligands have been developed as neuroimaging agents, including 11C-PK11195 in a positron emission tomography (PET) study of Parkinson’s disease dementia (10). In addition, previous work in mice has shown that TSPO ligands may be a potential therapeutic intervention to slow the progression or even reverse AD neuropathology (11). Among them, PK11195 and Ro5-4864 are commonly used in the research field of brain related cognitive function, and have been proved to have pleiotropic neuroprotective effect. The TSPO ligand isoquinoline carboxamide (PK11195) previously has been shown to decrease microglial activation and inflammation (12). PK11195 can specifically bind to TSPO, but Ro5-4864 needs the assistance of other mitochondrial proteins to complete the binding of TSPO protein. In animal models of neurodegenerative diseases (13) and neuroinflammatory psychosis (14), TSPO ligands have been shown to have significant in vivo efficacy. These studies emphasized that TSPO ligands can be used for neuroprotection, limiting neuroinflammation, promoting glial cell regeneration, and treating nervous system dysfunction.

At present, studies on the relationship between the occurrence of aMCI during aging and the up-regulation of TSPO expression, as well as the potential preventive or therapeutic role of TSPO classic ligands in aMCI, are lacking. Age-associated increases in the expression of TSPO in the central nervous system (CNS) may affect the astrocytes accompanying CNS development. As supporters of important neurogenic physiological functions, astrocytes perform four main functions: (I) supporting and isolating the original structure of the central nervous system, such as neural tubes, in the embryonic period (15); (II) adjusting the concentration of internal and external ions and transmitting the second messenger; (III) ingesting, inactivating, and supplying neurotransmitters (16); and (IV) regulating nutritional repair and antioxidant effects (17,18). To ensure normal neurophysiological function, the maintenance of the biological integrity of astrocytes are of great significance. Previous studies have confirmed that the mitochondria is the core structure involved in the regulation of apoptosis. Under the stimulation of various damage factors, the mitochondria can undergo osmotic translocation (19), eventually leading to the transfer of apoptotic proteins from the mitochondrial bilayer membrane into the cytoplasm (20).

Our previous study showed that subcutaneous injection of D-gal (1,000 mg/kg/day) in rats can result in mild nerve pathological changes, cholinergic system function impairment, mild oxidative damage, and abated oxidation resistance, as well as slight decreases in spatial learning and memory abilities, with little impact on motivation and foraging behavior. Therefore, in order to explore the mechanism of the TSPO-induced pathophysiology of astrocyte morphology, we chose to induce brain aging in rats using the classic method of continuous injection of D-gal via the back of the neck; at the same time, the rats were administered repeated injections of TSPO ligand PK11195 via the intraperitoneal cavity to observe its effect on D-gal-induced cognitive function in aMCI.

We present the following article in accordance with the ARRIVE reporting checklist (available at http://dx.doi.org/10.21037/atm-20-6157).

Methods

Ethical statement

Experiments were performed under a project license (No.: 2018-321) granted by Fujian experimental animal association, in compliance with EU Directive 2010/63/EU for the care and use of animals.

Animals

Forty male Sprague-Dawley rats (age, 10–12 weeks; weight, 280±50 g) were purchased from Shanghai Slake Experimental Animal Co., Ltd. [qualification certificate no.: SCXK (Shanghai) 2017-0005]. The animals were kept at the Experimental Animal Center of Fujian Medical University [SPF (specific-pathogen-free) level, license number: SYXK (Min) 2016-0006]. The rats were subjected to cognitive function behavioral tests in The Anesthesiology Laboratory of the First Affiliated Hospital of Fujian Medical University. The animals were given free access to food and water, under a 12-h light (6:00–18:00)/dark (18:00–6:00) cycle. The feeding room was kept at a constant temperature (20–25 °C) and humidity (55%). The rats were given a 5-day adaptation period before the experiment.

Groups

Rats were randomly assigned to the following groups according to body weight: the control group (n=10), the D-gal group (aMCI group, n=10), the PK11195 + D-gal group (n=10), and the PK11195 group (n=10). The PK11195 + D-gal and PK11195 groups were intraperitoneally injected with PK11195 (3 mg/kg)(11). Starting 3 days after the PKIII95 injection, the rats in the D-gal and PK11195 + D-gal groups were administered an injection of D-gal (1,000 mg·kg^-1·D^-1) via the head and neck for 7 consecutive days. The D-gal was dissolved in saline, and PK11195 was dissolved in saline containing 0.4% Tween 80 (flow chart, Figure 1).

Figure 1 Experimental flow chart. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose; MWM, Morris water maze; HE, hematoxylin-eosin staining; IF, immunofluorescence.

Behavioral testing

The Morris water maze (MWM) was used to test for behavioral changes in the model rats. The MWM tests the learning and memory ability of rats based on their spatial positioning and sense of direction by forcing them to find a transparent platform hidden under the water surface. The 5-day water maze navigation experiment was started 1 day after the end of building mode, and the space exploration experiment was conducted 1 day after the completion of navigation experiment.

Pathology testing

After the behavioral test, four rats from each group were selected at random. They were fasted for 12 hours but could drink water freely. The mice were then sacrificed, their brains were removed, and the brain tissues were immersed in the fixed solution for 24 h and then embedded in paraffin the next day. After confirming the anatomic location of hippocampus, serial sections were taken on coronal plane. Some sections were stained with hematoxylin eosin. The other part of the slices were immersed in silver solution, and then treated with reducing agent, the silver particles settled in the axonal plasm, making them black brown.

Immunofluorescence (IF)

Four rats were randomly selected from each group. They were fasted 12 hours but had free access to water. The mice were then sacrificed, and the brain tissue was collected and embedded in paraffin. Some paraffinized sections were randomly selected for dewaxing in water. The primary antibody was PBR antibody (Abcam, ab92291) with dilution ratio of 1:100; the secondary antibody was Cy3 antibody (Servicebio, gb21404) with dilution ratio of 1:200. After fluorescent staining, the slices were observed under a fluorescence microscope and the images were collected (the 4',6-diamidino-2-phenylindole (DAPI) ultraviolet (UV) excitation wavelength: 330–380 nm, emission wavelength: 420 nm, blue light; fluorescein isothiocyanate (FITC) excitation wavelength: 465–495 nm, emission wavelength: 515–555 nm, green light; CY3 excitation wavelength: 510–560 nm, emission wavelength: 590 nm) .

Statistical analysis

Statistical analyses were performed with IBM SPSS STATISTICS 22.0(Armonk, NY: IBM Corp). The data are presented as mean ± standard error. The data obtained from the MWM localization and navigation experiment during the escape latency period were analyzed using the repeated measures analysis of variance (ANOVA). One-way ANOVA was applied for the data from space exploration experiments and the results of immunofluorescence detection. The data of each group were first tested for normality and homogeneity of variance. Two-way ANOVA was used for the measurement data of body weight and swimming speed. The Bonferroni post-hoc test was used to explore differences between groups. P<0.05 was considered statistically significant.

Results

Animal characteristics

Weight changes in rats

The rats in each group showed good activity and normal food and water consumption; none of the rats in these groups died. In each group, the weights of the rats in the early, middle, and late stages (i.e., days 1, 3, 5, and 7) of the drug intervention period conformed to a normal distribution. There were no statistically significant differences in the body weight of the rats on day 1, 3, 5, or 7 (Table 1, Figure 2, P>0.05).

Table 1 Comparison of body weight (g) of the rats during the experiment ($\overline{\chi }\pm s$, n=10)
Full table

Figure 2 Weight comparison during the experiment ($\overline{\chi }\pm s$, n=10). aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Changes in swimming speed

The typical measurement results in the early, middle, and late stages (days 1, 3, and 5, respectively) were selected for statistical analysis. The swimming speeds of the rats in each group on days 1, 3, and 5 were normally distributed. The results of the water maze experiment showed no statistically significant difference in the swimming speed of the rats in each group on day 1, 3, or 5 (Table 2, Figure 3, P>0.05).

Table 2 Comparison of swimming speed (mm/s) between the groups of rats ($\overline{\chi }\pm s$, n=10)
Full table

Figure 3 Swimming speed (mm/s) comparison ($\overline{\chi }\pm s$, n=10). aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Place navigation test

Rat trajectory changes

In the early stage of the MWM positioning experiment, compared with the control group, the aMCI group’s behavior of circumventing the wall was more obvious, which was mainly represented by the extension of the movement path and the reduction of the number of crossings in the middle area of the quadrant. In the early stage, the model rats pre-treated with PK11195 occasionally traversed the middle area of the quadrant. In the middle and late stages, the PK11195 group could quickly find the platform, and the behavior path was similar to that of the control group, while the PK11195 + D-gal group’s motion path was longer than that of the control group. Meanwhile, compared to the aMCI group, the PK11195 group took a shorter time to find the platform and crossed the middle area of the quadrant more times, with a straighter and single trajectory. During the experiment, the main movement modes were: “edge type → random type → trend type” in the aMCI group; “trend type → linear type” or “random type → trend type → linear type” in the control group and the PK11196 group; and “edge type → random type” or “random type → trending type → linear type” in the PK11195 + D-gal group (Figure 4).

Figure 4 Trajectories of the groups in the water maze navigation experiment. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Changes in rat escape latency

The results of the escape latency test differed between the groups (Table 3, Figure 5, F=11.661, P<0.001). Multiple comparisons [least significant difference (LSD) t-tests] showed that there were no statistically significant differences between the control group and the PK11195 + D-gal group (P>0.05). Compared with the control group, the aMCI group had prolonged escape latency (t=−5.337, *P<0.05). Moreover, the escape latency of the PK11195 group was shorter than that of the control group (t=2.255, *P<0.05).

Table 3 Comparison of escape latency between the groups of rats ($\overline{\chi }\pm s$, n=10)
Full table

Figure 5 Comparison of escape latency in the groups of rats ($\overline{\chi }\pm s$, n=10). *, compared with the control group, P<0.05; ^#, compared with the aMCI group, P<0.05. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Spatial probe test

The number of platform crossings was lower in the aMCI group than in the control group (*P<0.05); however, there was no significant difference with the number of platform crossings in the PK11195 group (P>0.05). The PK11195 + D-gal group had a higher number of platform crossings than the aMCI group (^#P=0.019, ^#P<0.05) (Table 4, Figure 6).

Table 4 The number of platform crossings by each group of rats ($\overline{\chi }\pm s$, n=10)
Full table

Figure 6 The number of platform crossings by each group of rats ($\overline{\chi }\pm s$ n=10). *, compared with the control group, P<0.05; ^#, compared with the aMCI group, P<0.05. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Pathological changes in hippocampal CA1 area of rat brain

HE staining

Histological appearance of pyramidal cells at low magnification

Figure 7 Pathological changes in pyramidal cells in the CA1 region (10×10). (A,B,C,D) represent the pathological changes of the pyramidal cells (blue-purple) in the hippocampal CA1 region of the rats from different experimental groups under 10×10 magnification. (A) is the control group, (B) is the aMCI group, (C) is the PK11195 group, and (D) is the PK11195 + D-gal group. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Histological appearance of pyramidal cells at high magnification

Figure 8 Morphological changes of pyramidal cells in CA1 region (40×10). (A,B,C,D) represent the pathological changes of the pyramidal cells (blue-purple) and intracellular structure of the hippocampal CA1 region of the different experimental groups under 40×10 magnification. (A) is the control group, (B) is the aMCI group, (C) is the PK11195 group, and (D) is the PK11195 + D-gal group. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Silver staining

Histological appearance of astrocytes at low magnification

Figure 9 Pathological changes of astrocytes in gray matter near CA1 region (10×10). (A,B,C,D) represent the pathological changes of astrocytes (black-brown) in the gray matter of the hippocampus in the different experimental groups under 10×10 magnification. (A) is the control group, (B) is the aMCI group, (C) is the PK11195 group, and (D) is the PK11195 + D-gal group. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Histological appearance of astrocytes at high magnification

Figure 10 Pathological changes of astrocytes in gray matter near CA1 region (40×10). (A,B,C,D) represent the pathological changes of astrocytes (black-brown) in the gray matter near the hippocampal CA1 region of the different experimental groups under 40×10 magnification. (A) is the control group, (B) is the aMCI group, (C) is the PK11195 group, and (D) is the PK11195 + D-gal group. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Distribution of TSPO expression by immunofluorescence

The expression and location of TSPO in the hippocampus of the aMCI group rats, along with the effect of PK11195 on TSPO protein expression, were further investigated by immunofluorescence (Figures 11,12). In the aMCI group, a large amount of TSPO protein was expressed in the pyramidal cell membrane in the hippocampal CA1 region of the brain, which showed bright red fluorescence. At least three 200-fold fields of vision were randomly selected for each slice in each group for semi-quantitative analysis (Figure 13). Compared with the control group, the expression of TSPO in the hippocampal CA1 region of the aMCI group was significantly higher than that in (F=30.547, ***P<0.001). However, the expression of TSPO protein around the pyramidal cell membrane in the hippocampal CA1 area of the PK11195 + D-gal group was not statistically different from that in the control group (P>0.05), and the expression of TSPO protein in the PK11195 group was not statistically different from that in the control group (P>0.05).

Figure 11 Coronal scans of slices of rat brain stained by immunofluorescence. (A,B,C,D) represent the control group, aMCI group, PK11195 group, and PK11195 + D-gal group respectively. The nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) blue and TSPO with red staining. The TSPO expression area of brain tissue in the aMCI group was wide and the fluorescence density was high; the expression range of TSPO in aMCI rats after PK11195 pretreatment became narrower and the fluorescence density decreased. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Figure 12 Immunofluorescence scan of the hippocampal CA1 region (40×10). The effect of PK11195 on TSPO expression and location in the hippocampal CA1 region. The nuclei were stained with DAPI (4',6-diamidino-2-phenylindole)blue and TSPO with red staining. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Figure 13 Analysis of TSPO expression in the hippocampal CA1 region ($\overline{\chi }\pm s$, n=4). ***, compared with the control group, P<0.001. aMCI, amnestic mild cognitive impairment; D-gal, D-galactose.

Discussion

TSPO-related physiological functions have mainly been studied through TSPO ligands, which are closely related to the occurrence and development of central neurodegenerative diseases. TSPO ligands are mainly divided into endogenous and exogenous ligands, among which cholesterol and porphyrins are significant. Aβ protein is known to play a major pathological role in the brains of AD patients; astrocytes can induce endogenous TSPO ligands after induction by Aβ protein (21), which means endogenous TSPO ligands can be used as biomarkers. Various neuropathological studies have been conducted on brain aging and reactive glial hyperplasia. In addition to the pathological diagnosis of endogenous ligands, recent studies have shown that exogenous TSPO ligands can be used for neuroprotection, as well as to limit neuroinflammation, promote glial cell regeneration, and treat nervous system dysfunction (22). Among these exogenous ligands, PK11195 and Ro5-4864 have been shown to exert multiple neuroprotective effects and have been commonly used in the field of brain-related cognitive function research (23). PK11195 can specifically bind to TSPO, whereas Ro5-4864 requires the assistance of other mitochondrial proteins (24). In view of this difference in binding function between the two ligands, this experiment used PK11195 to conduct a more direct and concise mechanism study.

Previous studies confirmed that mitochondrial damage is an important initiating factor leading to cell apoptosis (25). The dysfunction of mitochondrial damage in relevant nerve cells in the brain leads to nerve cell damage in the learning and memory brain region, and eventually leads to cognitive dysfunction. When D-gal and other pro aging factors lead to astrocyte damage, the increase of TSPO protein expression in the local area will lead to the disorder of mitochondrial cholesterol synthesis, which will affect the function of mitochondrial synthesis and metabolism, and then lead to a large number of apoptotic substances such as AIF, Cyt C, and release to the cytoplasm, and finally accelerate the apoptosis of astrocyte. Previous studies have shown that excessive activation of glial cells (mainly microglia) in the process of brain injury leads to a large number of TSPO expression (26), and ultimately accelerates cognitive impairment. With the accumulation of TSPO, mitochondria in astrocyte release apoptosis related inducing factors, which changes the structure and biological function of astrocyte. After TSPO is activated by aging factors, the transport of TSPO dependent cholesterol to mitochondria is accelerated, which results in the impairment of cholesterol synthesis function and further leads to neurological dysfunction

The immunofluorescence results of this study showed that the expression areas of TSPO in the hippocampal CA1 region of rat hippocampus tissue after PK11195 pretreatment were narrower than that in aMCI group. During the experiment, the body weight of the rats did not fluctuate significantly during intervention with PK11195, nor did the swimming speed of the rats change abnormally, which indicates that PK11195 did not affect the normal limb motor function of the rats. During the behavior test, the ability of the rats in the control, PK11195, and PK11195 + D-gal groups to find the platform increased continuously with training time, and the time required to locate the platform became shorter over time, indicating that PK11195 effectively relieved D-gal-induced learning and memory dysfunction. Compared with the rats in the aMCI group without intervention, the number of times the rats in the PK11195 + D-gal group traversed the platform increased, indicating that their spatial recognition and memory function had recovered; meanwhile, compared with the normal rats, the number of platform crossings in the PK11195 group did not increase, suggesting that injection of PK11195 can alleviate the mild learning and memory function decline caused by D-gal without affecting the normal memory and learning ability of the brain. The pathological results showed that PK11195 can prevent the pathological changes of pyramidal cells caused by D-gal, maintain the normal morphological structure of pyramidal cells, and delay the processes of cell damage and apoptosis caused by D-gal. Immunoblotting experiments further revealed that the expression of TSPO in the hippocampus of rats in the PK11195 + D-gal group was lower than that in the aMCI group. The expression of TSPO is generally believed to significantly increase with AS damage or microglia activation in brain tissue and can be used as a sensitive indicator to judge the functional integrity of mitochondria in the brain. Compared with the rats in the aMCI group, the expression of TSPO in the hippocampus of rats in the PK11195 + D-gal group was significantly decreased, confirming that PK11195 can significantly inhibit the D-gal-induced expression of TSPO protein in the hippocampus. Furthermore, compared with the normal rats, no significant difference was observed in TSPO expression in the hippocampus of the PK11195 group rats, suggesting that PK11195 alone did not affect the normal physiological expression of TSPO.

Conclusions

The findings of the present study can be summarized as follows: (I) the exogenous TSPO ligand PK11195 can alleviate D-gal-induced cognitive impairment associated with early learning and memory function; (II) PK11195 can prevent the pathological changes of astrocytes and pyramidal cells by reducing the high expression of TSPO induced by D-gal in the hippocampus, and alleviate early decline in learning and memory function. It has been shown that voltage dependent anion channel (VDAC) and mitochondria inner membrane protein adenine nucleotide translocator (ANT) constitute the core of mitochondrial multiprotein complex (MPTP) (27). Since TSPO is located in the outer membrane of mitochondria and is associated with VDAC, it suggests that TSPO may be related to MPTP; at the same time, the loss of mitochondrial endomembrane potential in response to apoptosis induction can be prevented by directly regulating TSPO to regulate MPTP complex (28). The mechanism of PK11195 in preventing as induced by D-gal may involve the change of binding function between TSPO and MPTP.

In this experiment, the expression of TSPO in the hippocampus of the aMCI group was found to be abnormally increased. At the same time, the results of immunofluorescence showed that D-gal induced early decline in learning and memory function along with abnormal expression of TSPO in the hippocampus, and other brain areas also showed increased expression of TSPO. Our results suggest that expression of TSPO in the brain tissue of amnestic cognitive impairment is increased, which can lead to cognitive behavior abnormalities

Acknowledgments

Funding: This work was supported by the Natural Science Foundation of Fujian Province, China (Grant No. 2018J01178) and the Project of Fujian Medical Innovation (Grant No. 2015-CX-22).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at http://dx.doi.org/10.21037/atm-20-6157

Data Sharing Statement: Available at http://dx.doi.org/10.21037/atm-20-6157

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-6157). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No.: 2018-321) granted by Fujian experimental animal association, in compliance with EU Directive 2010/63/EU for the care and use of animals,

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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