Animals

Animal procedures were approved by the AEC (animal ethics committee) of HKUST (Hong Kong University of Science and Technology), identified by the approval number AEP-2022-0038 titled “Study on Gabrb2 associated functional and behavioral effects using mouse model”. All experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Gabrb2-KO mice (hybrid of C57BL/6-129/SvEv) and WT control mice of both sexes [16] were bred and grown at the in-house animal facility until they were more than 7 months old, with the majority more than 12 months old and several of them more than 24 months old.

Mouse genotyping

Ear punching was used to collect mouse tissue for genotyping to confirm the genotypes of the KO and WT mice. Alkaline lysis (75 µL) buffer was added to the tissue, heated for 1 h at 95°C, and shaken for 30 min. The mixture was cooled to 4°C and neutralized; a 5 µL aliquot was employed for PCR amplification using specific primers for the Neo (neomycin resistance gene) and exon 7 of the Gabrb2 gene (Table 1). Amplified products were centrifuged for 10 s, then subjected to 2% agarose gel electrophoresis to determine the genotypes.

Herbal extract preparation

The herbal combination was prepared from Radix Bupleuri chinense DC, Rhizoma Corydalis yanhusuo, Caulis Polygonum multiflorum, and Flos Albizia julibrissin. In brief, the herbs were finely powdered and boiled for 2 h in 2 L of 5% acetic acid twice. The supernatants were collected, filtered, reboiled, and dried at 92°C. Ethanol (100%) was added to the powder and reboiled and redried at 92°C. WT-aged mice were separated into a vehicle (Veh) group receiving a daily oral administration of 0.9% NaCl for 4 weeks, and a herbal group receiving, additionally, a daily oral administration of BYPA in 0.9% NaCl (60 mg/kg).

Behavioral tests

Behavioral tests were performed on aged WT and Gabrb2-KO mice to examine whether Gabrb2-KO phenotypes observed in young adult mice would remain in aged mice, and also performed on aged WT mice for both Veh and BYPA groups to search for the anti-aging effects in naturally aged mice.

Hole-board test

The hole-board consisted of a 60 × 60 cm square-shaped floor with 20 cm high walls on each side. Four holes with 3 cm diameter were evenly distributed on the floor. Each mouse was placed at the center of the floor at the start of the test, and allowed to roam for 5 min, during which the number of head-dips into the holes and the number of climbs onto the walls were counted. This test assessed the anxiety-like behavior of the mouse and the sedative effect of any drug administered to it. A decrease in the frequency of climbs or head-dips signified a sedative effect by the drug [31].

Three-chamber test

The three-chamber test was used to assess social affiliation (or sociability) and preference for social novelty [32]. The apparatus consisted of a rectangular-shaped transparent box with three chambers (a central chamber and two side chambers) and openings between the chambers. The chambers were 19 cm × 45 cm in dimension, and the openings between the chambers could be closed off using a removable plexiglas partition. An upside-down pencil cup was placed inside each side chamber to hold a stranger mouse. Before the test, the test mouse was positioned in the central chamber and allowed to roam for 5 min to familiarize itself with the apparatus. A stranger-1 mouse with no previous contact with the test mouse and similar in age was placed inside the cup in one of the side chambers. The plexiglass partitions between the three chambers were removed for 10 min. We measured the social affiliation in terms of the number and total duration of the visits exhibited by the test mouse for the cup containing the stranger-1 mouse relative to the empty cup. Thereupon, a novel stranger-2 mouse was placed in the cup in the other side chamber, and the number and total duration of visits by the test mouse to the novel stranger-2 mouse relative to the now familiar stranger-1 mouse were recorded in order to determine the preference of the test mice for social novelty.

Locomotor activity test

A plastic, transparent circular container that was exposed at the top was employed in this test. Three strips of tape were fixed to the bottom of the transparent container to create six equal segments of the circle. The locomotor movement of a mouse across different segments was measured by the frequency of its tail crossing between any two segments during a 5-minute test period. If its tail crossed a segment while the mouse was rotating on a spot within the same segment, it would not be counted.

Elevated plus maze test

The elevated plus maze consisted of a “+”-shaped elevated platform with four arms and a central square platform. Two opposite arms were closed arms with walls, while the remaining two were open arms without walls. The maze was elevated to 40 cm above ground. At the start, the mouse was positioned on the central platform and allowed to move around the maze for 5 min. During this period, its entries into the closed and open arms were counted and timed separately, while entries into the central platform were not counted. Increased frequency or duration of visits to the open arms over the closed arms signified an anxiolytic effect [33].

Tail suspension test

The tail suspension test was used to detect depression-like behavior. The apparatus consisted of a box with a horizontal bar set 15 cm above the floor. The mouse was taped by its tail to the horizontal bar and subjected to 6 min of hanging. The immobility time within the 6 min was recorded. This test assessed the effects of a drug on the level of the mouse’s depression, and a decrease in immobility time caused by the drug testified to the antidepressant effect of the drug [34].

Oxidative stress analysis (MDA)

Mice were euthanized using cervical dislocation, which was approved by the AEC. The brain of WT-aged mice (Veh and BYPA groups) was removed and homogenized in phosphate buffer saline, pH 7.2. Oxidative stress was estimated by the reaction between TBA (thiobarbituric acid) and MDA using the spectrophotometric assay at 532 nm [35].

Gene expression analysis

The analysis of the expression of antioxidant enzyme genes in the brain of WT-aged mice (Veh and BYPA groups), including Sod2 (superoxide dismutase) and Cat (catalase), was performed by RT-qPCR. Total RNA (ribonucleic acid) was extracted using TRIzol reagent (Invitrogen Corp, CA, 15596026) according to the manufacturer’s instructions. Total RNA was reverse transcribed using QuantiTect^® reverse transcription kit (Qiagen, 4368814). RT-qPCR was duplicated for each sample using FastStart Universal SYBR Green Master (Roche, 45-4913850001). ACTB (Beta-Actin, 4333762F) was used as a reference. The primers used in these experiments are described in Table 1.

Statistical analysis

Results were expressed as mean ± SD. All statistical analyses were performed using Prism 8.0 (Graphpad Software), and statistical significance was established by p < 0.05. Data were analyzed by means of a one-way ANOVA test followed by Tukey’s post hoc test or student’s unpaired t-test.

Behavioral changes in aged Gabrb2-KO mice

A spectrum of schizophrenia-like behavioral changes was observed in young adult Gabrb2-KO mice relative to WT mice [16]. In the present study, the aged Gabrb2-KO mice also displayed an increased number of stereotypic climbs in the head-dip test, although not in the number of stereotypic head-dips (Figure 1A).

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Figure 1. Behavioral tests on aged WT and Gabrb2-KO mice. A) Hole board test: The number of head-dips into the circles and the number of climbs on the walls were counted during a 5-minute interval (WT, n = 14; KO, n = 14). B) Social affiliation: The assessment of both time and number of visitations to the stranger-1 mouse for a 10-minute interval (WT, n = 6; KO, n = 6). C) Social novelty: The assessment of both time and number of visitations to the stranger-2 mouse for a 10-minute interval (WT, n = 6; KO, n = 6). D) Locomotor activity test: The movement of the mouse across each segment was measured by counting the number of times its tail crossed a segment for a total of 5 min (WT, n = 14; KO, n = 14). E) Elevated-plus maze test showing percentile time spent in or entries into open arms (WT, n = 6; KO, n = 6). F) Tail suspension test where the mouse was taped by the tail to the horizontal bar and subject to 6 min hanging. The immobility time was recorded within 6 min (WT, n = 14; KO, n = 14). WT mice are shown in blue and KO mice in green. Statistical analysis was performed using student’s t-test for locomotor activity and tail suspension tests and using one-way ANOVA with Tukey’s post hoc test for other behavioral tests. Data are expressed in mean ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001. Average age (WT = 15.5 months, KO = 15.1 months).

In the three-chamber test, the KO mice did not differ significantly from the WT mice in the number or duration of visits to the cup containing the stranger-1 mouse relative to the empty cup without any stranger mouse (Figure 1B). There was no significant difference in the number or duration of visits to the cup containing the novel stranger-2 mouse relative to the cup containing the stranger-1 mouse, which had become the familiar mouse (Figure 1C). Therefore, Gabrb2-KO did not affect significantly either the social affiliation of the test mice or the extent of their preference for social novelty behavior.

In the locomotor activity test, the Gabrb2-KO mice showed a much higher level of activity than the WT mice (Figure 1D), plausibly as a manifestation of their psychotic agitation.

In the elevated plus maze test, the Gabrb2-KO mice showed an elevated number and total duration of entries into the open arms compared to the WT mice (Figure 1E), indicative of diminished anxiety in the KO mice. In the tail suspension test, the Gabrb2-KO mice showed reduced immobility time compared to the WT mice (Figure 1F), suggesting that the KO mice were less susceptible to depression than the WT mice.

Effects of BYPA on behavior of aged mice

The BYPA-treated group received a daily oral administration of BYPA for 4 weeks, and the Veh group of animals received daily oral administration of vehicle (0.9% NaCl). Daily oral treatment with BYPA significantly decreased locomotor activity relative to the control mice receiving only administration of vehicle with p < 0.01 (Figure 2A). In the elevated-plus maze test for anxiety, a significant increase in the number of entries into open arms in the elevated plus maze test with p < 0.05 (Figure 2B), which indicates a decreased level of anxiety. In the tail suspension test to assess depression level, a significant decrease in immobility time was observed with p < 0.05 (Figure 2C), indicating a reduced level of depression relative to WT.

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Figure 2. Behavioral tests for BYPA herbal formula treatment on aged WT mice. A) Locomotor activity test: The movement of the mouse across each segment was measured by counting the number of times its tail crossed a segment for a total of 5 min (Veh, n = 10; BYPA, n = 9). B) Elevated-plus maze test showing percentile time spent in or entries into open arms (Veh, n = 11; BYPA, n = 9). C) Tail suspension test where the mouse was taped by the tail to the horizontal bar and subject to 6 min hanging. The immobility time was recorded within 6 min (Veh, n = 10; BYPA, n = 9). Veh group mice are shown in blue and BYPA group mice in green. Statistical analysis was performed using student’s t-test for locomotor activity and tail suspension tests and using one-way ANOVA with Tukey’s post hoc test for other behavioral tests. Data are expressed in mean ± SD. * p < 0.05, ** p < 0.01. Average age (Veh = 16.7 months, KO = 15.8 months).

Effects of BYPA on oxidative stress and antioxidant enzymes

In the young adult Gabrb2-KO mice, the brain level of oxidative stress measured by MDA was enhanced, and the levels of the inflammatory cytokines TNF-α and IL-6 were also elevated, suggesting that the schizophrenic-like phenotypes of these mice were associated with an oxidant/antioxidant imbalance skewed toward an excessively oxidative status [16].

Since aging is characterized by inflammation with ROS (reactive oxygen species), resulting in an inflammaging process plausibly arising from immuno-decay [36, 37], the phenotypic overlap of inflammation and excessive oxidation between the disorders of schizophrenia and aging is striking. Accordingly, in view of the anti-aging activities of BYPA on mice under accelerated aging through galactose treatment [24], the effects of BYPA on naturally aged mice were analyzed in Figure 3. The results showed that BYPA reduced the brain level of MDA (Figure 3A) and enhanced the expression of Cat (Figure 3B), both to p < 0.05.

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Figure 3. MDA analysis and mRNA expression of antioxidant enzymes in aged WT mice. A) MDA analysis: measured by the reaction between TBA and MDA and expressed per mg protein of sample analyzed (Veh, n = 11; BYPA, n = 11). B) mRNA expression for Sod2 and Cat in the brain was measured using RT-qPCR (Veh, n = 12; BYPA, n = 12). Veh group mice are shown in blue and BYPA group mice in green. Statistical analysis was performed using student’s unpaired t-test for the MDA test and one-way ANOVA with Tukey’s post hoc test for RT-qPCR. Data are expressed in mean ± SD. * p < 0.05. Average age (Veh = 16.7 months, KO = 15.8 months).

Hair regeneration

Some of the aged WT-mice showed facial hair loss before the start of a test for facial hair regeneration. Hair regrowth was checked constantly to confirm our previous results on galactose-induced aging mice [24]. Importantly, the BYPA group displayed facial hair regeneration after four weeks of daily treatment in contrast to the Veh, which showed no significant facial hair regeneration (Figure 4).

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Figure 4. Facial hair regeneration in the BYPA group. A) Photographs of a Veh group mouse (A1) before treatment and (A2) after 4 weeks of treatment with vehicle. B) Photographs of a BYPA group mouse (B1) before treatment and (B2) after 4 weeks of treatment with BYPA.

Effects of aging on the behavior of KO mice

The present study examined behavioral symptoms of aged Gabrb2-KO mice in comparison to WT mice using behavioral tests, including social inclinations, and also affective tests for anxiety and depression. The findings of several differences between the aged KO mice and normal aged mice have confirmed the problem posed by old-age schizophrenia, including neuroinflammation with increased pro-inflammatory cytokines, as well as increased oxidative stress, and microglial activation, together with hyperactivity, decreases in cell proliferation in the hippocampus, and epilepsy [16]. Moreover, we previously confirmed that Gabrb2 was associated with schizophrenia [38–40] and was subjected to altered methylation, confirming epigenetic regulation of its expression [11]. Epigenetic changes promote accelerated aging and persisting schizophrenia symptoms in Gabrb2-KO mice [38]; all these findings pointed to the plausible utility of aged KO mice as animal models in this regard.

Gabrb2-KO mice exhibited behavioral and cognitive changes similar to those observed in schizophrenia, including: Neuroinflammation with increased oxidative stress, increased pro-inflammatory cytokines, and microglial activation, as well as the comorbidities of hyperactivity, decreases in cell proliferation in the hippocampus, and epilepsy [16].

Although schizophrenic patients often experience a moderation of some of their disturbing symptoms, old-age schizophrenia has emerged as a highly distressing disease. While the aged manifest fewer positive symptoms, their negative and cognitive symptoms tend to persist. It is estimated that old-age schizophrenia in New Hampshire is twice as expensive to manage as depression and three times as difficult to manage as a physical disease. There are also two apparent sources of old-age schizophrenia, with 80–85% early-onset patients developing the disease before age 60, and 15–20% late-onset patients developing the disease after age 60 [22]. It is unclear whether these two groups differ only in the timing of the occurrence of their symptoms or in the mechanisms of their etiology as well.

Protective effects of BYPA on a natural aging model

The encounter of oxidative stress in aging [40] and in Gabrb2-KO mice [16] represents a striking co-occurrence, suggesting that there could be a mechanistic link between aging and schizophrenia in accord with the view that aging might be a disorder, such as an oxidative or immunological disease [36, 37]. BYPA, which is a combination of four potential medicinal herbs based on traditional Chinese medicine (TCM), showed anti-aging and anxiolytic effects as well as reduced oxidative stress and neuroinflammation in an induced aging model with no significant side effects [24]. The present study brought about anti-aging changes also among the naturally WT-aged mice. BYPA lowered the brain level of MDA and increased the expression of anti-inflammatory enzymes (Cat). Increased oxidative stress is common in many neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [39, 40]. BYPA may be a useful therapy to prevent or delay the occurrence of aging-related diseases. BYPA extracts showed significant anxiolytic and sedative effects, with repair of facial skin and regrowth of facial hair. In this regard, BYPA is suggested to be a potential treatment for insomnia and anxiety [28]. All these findings are supportive of the usage of BYPA as an anti-aging health supplement for the aged population, including aged schizophrenics.

The present study carried out behavioral experiments with two genotypes of the same strain of mice to provide insights into aging and anti-aging from different perspectives. On one hand, behavioral experiments with naturally aged Gabrb2-KO mice demonstrated that the behavioral effects of genetic defects persisted, providing a stable mouse model for relevant neuropsychiatric pharmacology studies, including anti-neuroinflammation, since Gabrb2-KO has been shown by us previously to induce extensive neuroinflammation in mice. On the other hand, BYPA, an anti-neuroinflammatory herbal formula previously reported by us [24], has been surprisingly demonstrated herein with anti-aging effects on hair protection and recovery. BYPA has been strongly recommended to be administered to naturally aged Gabrb2-KO mice. However, compromised fertility in naive KO mice and the long period of mice breeding and needing to orally administer BYPA for 4 weeks after behavioral tests, together with their susceptibility to audiogenic epilepsy, make them fragile and incapable of accomplishing the experiment.

Knowledge generated from this study paves the way to the development of useful anti-neuroinflammation and anti-aging therapeutics and nutraceuticals to address the needs of rapidly aging societies.