Photobiomodulation Therapy Attenuates Anxious-Depressive-Like Behavior in the TgF344 Rat Model

Abstract

Background:

Anxious-depressive-like behavior has been recognized as an early endophenotype in Alzheimer’s disease (AD). Recent studies support early treatment of anxious-depressive-like behavior as a potential target to alleviate memory loss and reduce the risk of developing dementia. We hypothesize that photobiomodulation (PBM) could be an effective method to alleviate depression and anxiety at the early stage of AD pathogenesis.

Objective:

To analyze the effect of PBM treatment on anxious-depressive-like behavior at the early stage of AD.

Methods:

Using a novel transgenic AD rat model, animals were divided into wild-type, AD+sham PBM, and AD+PBM groups. Two-minute daily PBM (irradiance: 25 mW/cm² and fluence: 3 J/cm² at the cortical level) was applied transcranially to the brain of AD animals from 2 months of age to 10 months of age. After completing PBM treatment at 10 months of age, behavioral tests were performed to measure learning, memory, and anxious-depressive-like behavior. Neuronal apoptosis, neuronal degeneration, neuronal damage, mitochondrial function, neuroinflammation, and oxidative stress were measured to test the effects of PBM on AD animals.

Results:

Behavioral tests showed that: 1) no spatial memory deficits were detected in TgF344 rats at 10 months of age; 2) PBM alleviated anxious-depressive-like behavior in TgF344 rats; 3) PBM attenuated neuronal damage, degeneration, and apoptosis; and 4) PBM suppresses neuroinflammation and oxidative stress.

Conclusion:

Our findings support our hypothesis that PBM could be an effective method to alleviate depression and anxiety during the early stage of AD development. The mechanism underlying these beneficial effects may be due to the improvement of mitochondria function and integrity and the inhibition of neuroinflammation and oxidative stress.

Keywords

Anxiety depression low-level laser therapy photobiomodulation TgF344 rats

INTRODUCTION

Alzheimer’s disease (AD) is the most common form of dementia and is characterized by irreversible cognitive deficits and impaired learning and memory [1]. As a major neurodegenerative disease, AD contributes to high pharmaceutical research costs and exacts a heavy medical and financial toll on both society and patients [1, 2]. Although numerous approaches and treatments focusing on relieving typical symptoms have been developed, and numerous studies continue, no currently approved medical treatment can cure AD or stop its progression [3]. In addition to typical AD pathology hallmarks, such as amyloid plaques, neurofibrillary tangles, and cognitive deficits, nearly half of patients with AD experience depression, and up to 71%of people with AD experience anxiety [4]. In fact, recent studies have recognized anxious-depressive-like behavior as an early sign of AD pathogenesis in animal models [2, 5]. Emerging evidence suggests that early treatment of depressive-like behavior could alleviate later memory loss, thereby presenting a target to reduce the risk of developing dementia [6].

Mitochondrial abnormalities have been found in numerous brain disorders, including AD, depression, and anxiety [7, 8]. As the primary source of energy in mammalian cells, mitochondria play a crucial role in neuronal survival and apoptosis [9, 10]. In AD, mitochondrial dysfunction is the primary source of reactive oxygen species (ROS) and contributes to the oxidative damage and neuroinflammation that induces neuronal damage and apoptosis [8, 11]. Mitochondrial dysfunction contributes to the development of depression and anxiety by impairing neurogenesis, neuronal transmission, and synaptic plasticity necessary for successful adaptation to stressful conditions [12, 13]. Therefore, approaches targeting mitochondrial dysfunction may represent a potential avenue for preventing depression, anxiety, and memory loss in the development of AD [8].

Photobiomodulation (PBM), also known as low-level laser therapy and light-emitting diode therapy, has emerged as an effective non-invasive strategy to improve neurological diseases and disorders [14 –20]. Serving as a mitochondrial-targeted approach, PBM confers protective effects against mitochondrial dysfunction, neuroinflammation, and oxidative stress in various brain disorders [8 , 22]. Although PBM post-treatment improved depression-like behaviors in another study using a mouse model of major depressive disorder [23], whether PBM protects against anxious-depressive-like behavior in the context of AD is still unclear. Therefore, the present work was designed to investigate the effects of PBM treatment on anxious-depressive-like behavior in a transgenic AD rat model and uncover potential underlying mechanisms.

METHODS

Animals and experimental design

Male TgF344 rats were used in the present work. The TgF344 rats with human amyloid beta (A4) protein (hAPP) gene and human presenilin 1 gene (PS1) were generated on a Fischer 344 background [24]. According to previous studies, Tg344 rats do not show cognitive impairment before 18 months old, but exhibit increased anxious-depressive-like behavior [2, 5]. As shown in Fig. 1A, the AD animals were randomly divided into the following three groups: 1) WT animals (n = 8), 2) AD animals (N = 12), and 3) AD animals with PBM treatment (N = 12). PBM was initiated in 2-month-old animals and ended at 10 months of age. As reported in a previous study, 2-min daily PBM treatment was applied on the top of the rat’s head with a diode laser at 808 nm 3 times/week for 8 months [25]. The laser setup used in this study was based on our previous work [25]. The irradiance each animal received was 350 mW/cm² (fluence: 42 J/cm²) for the scalp and 25 mW/cm² (fluence: 3 J/cm²) for the cortex. All rats were shaved before applying PBM treatment. The laser beam spot (808 nm) was positioned using a fiber guide light which allows it to target the scalp easily. The distance between the end of the beam fiber lens (Imeter 400μm multi model; adjustable focus) and the rat scalp was adjusted to 35 cm to generate a 1.5 cm² laser spot size on the scalp.

Fig. 1

Schematic diagram of the experimental design and device information.

After PBM treatment, several behavioral tests were performed to measure behavioral changes in 10-month-old AD rats with or without PBM treatment. Finally, brains were collected for future studies. The information regarding the PBM device is shown in Fig. 1A.

Barnes maze task

The Barnes maze task was performed to determine spatial learning and memory, as described in our previous studies [1, 16]. Briefly, the Barnes maze task was divided into three once-daily 3-min training trials (first 3 days) and a 90-s probe trial (the 4th day). During training trials on the first 3 days, the rats were placed at the center of an elevated circular platform with 18 holes around the periphery, surrounded by four walls with differing patterns. A black escape box was hidden under one hole. Rats are allowed to explore and find the hidden box. An ANY-maze software-controlled overhead video camera was applied to record rats’ movement traces, escape latency, and escape velocity. If the rats cannot find the hidden box within 3 min, the time spent to find the hidden box was recorded as 3 min. During the probe trial test, the hidden box was removed, and the animals were allowed to explore the platform freely for 90 s. The time spent in the target quadrant (the quadrant where the hidden box was formerly located) and the number of exploring errors were recorded and analyzed. The apparatus was cleaned with 70%ethyl alcohol between every test.

Sucrose preference test

The sucrose preference test was used to examine depressive-like behavior, as previously described [2]. In brief, the sucrose preference test was divided into four stages. In the first stage, animals were housed in the cages with a choice of two bottles of plain water for 2 days, followed by the second stage with 2%sucrose solution for another 2 days. Following this acclimation, rats underwent water deprivation for 24 h. Finally, the rats were allowed to freely access either plain water or 2%sucrose solution for 8 h. The sucrose water and plain water consumption were recorded, and sucrose preference was calculated as follows: sucrose solution consumption/(sucrose solution + plain water consumption)×100%.

Forced swim test

The forced swim was applied to measure depressive-like behavior in rodents, as previously described [2, 26]. Briefly, rats were placed in a transparent plastic cylinder filled with 30-cm deep, 24±1°C water for 6 min. The immobile time was recorded by ANY-maze video tracking system.

Elevated plus maze

The elevated plus maze, a widely accepted test of measuring anxiety in laboratory animals, was performed to detect anxiety-like behavior in AD animals [2, 26]. The elevated plus maze used in the current study was elevated 50 cm above the floor with two closed arms and two open arms. The two opposing closed arms were surrounded by 50 cm-high walls. During the test, the rats were placed at the intersection of the open arms and closed arms and allowed to explore freely for 5 min. Open arm entries and time spent in the open arms were recorded and analyzed by ANY-maze video tracking system. The plus maze was cleaned with 70%ethanol in between each test

Tail suspension test

The tail suspension test is a widely used behavioral test for detecting anxious-depressive-like behavior [27]. The animal’s tail was fixed on a suspension box during the test, suspending the rat 50 cm above the ground. The immobile time was recorded during the 6 min of testing.

Open field test

The open field test was performed as described by previous studies to measure anxiety-related behavior [2, 26]. The apparatus used in the open-field test was a box with a black wooden floor surrounded by 50 cm-tall walls (56×56×50 cm). During the test, animals were placed at the same corner of the box and allowed to explore the box freely. The time spent in the center zone (26×26 cm), the entries into the central zone, and the number of defecations left in the box were recorded and analyzed. The box was cleaned between each test with 70%ethanol.

Brain extraction and tissue preparation

Brain collection was performed as described previously [15]. In brief, the rats were sacrificed under deep anesthesia and transcardially perfused with ice-cold saline. Half of the brain was used for sectioning and was postfixed with paraformaldehyde (PFA) overnight, followed by cryoprotection using 30%sucrose. Coronal sections (25μm each) were prepared from each rat brain using a cryostat (Leica RM2155, Nussloch, Germany). Cortical tissue from the remaining hemisphere was quickly separated and frozen in liquid nitrogen for tissue homogenization. Total protein sample collection was prepared using a motor-driven Teflon homogenizer. Ice-cold homogenization buffer (50-mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], NaCl, β-glycerophosphate, Triton X-100), protease and phosphatase inhibitors were used for tissue homogenization according to a previous protocol [1]. Protein concentrations of cortex tissue samples were determined by Modified Lowry Protein Assay (Pierce, Rockford, IL).

F-Jade C and TUNEL staining

Neuronal degeneration and apoptosis were determined using F-jade C and TUNEL staining, respectively. In brief, 25-μm thick brain sections were incubated for 20 min with Fluoro-Jade C (AG325, Sigma-Aldrich) working solution followed by 5 washes with PBS-Triton X-100. After washes, the sections were mounted and coverslipped with a water-based mounting medium. TUNEL staining was applied to detect neuronal apoptosis as previously described by our laboratory [26, 28]. According to manufacturer instructions, brain sections were stained using a Click-iT Plus TUNEL assay kit (Thermo Fisher Scientific). Images of F-Jade C and TUNEL staining were captured by a Zeiss LSM700 Meta confocal laser microscope (Carl Zeiss).

Caspase activity assay

Caspase-3 and caspase-9 activities were determined using fluorogenic substrates, as described previously by the author [29]. Ac-DEVD-AMC and Ac-LEHD-AMC (AnaSpec, Fremont, California) were applied as substrates to measure caspase-3 and caspase-9, respectively. The protein homogenate was mixed with substrates and incubated with protease assay buffer for 1 h at 37°C. The fluorescence of free 7-Amino-4-methylcoumarin (AMC) was measured by fluorescence spectrophotometer (Perkin Elmer) with excitation at 360 nm and emission at 460 nm. Caspase activities were expressed as changes in fluorescent compared to the WT group.

Immunofluorescence staining and confocal microscopy

Immunofluorescence staining was performed as described in our previous studies [14, 15]. In brief, selected brain sections were incubated with 10%normal donkey serum in 0.1%Triton X-100 for 1 h at room temperature, followed by incubation with corresponding primary antibodies overnight at 4°C. The following primary antibodies were used in this study: anti-MBP, p-H2AX, Iba-1, Tom20 (Proteintech, Rosemont, IL, USA); anti-PSD95, SOD2 (Novus Biologicals, Littleton, CO, USA); anti-MAP2 (Thermo Fisher Scientific, Waltham, MA, USA) and anti-4-HNE (Abcam, Cambridge, UK). After three washes with 0.4%Triton X-100, the brain sections were then incubated with appropriate Alexa Fluor donkey anti-mouse/goat/rabbit secondary antibodies (488/549/647, Thermo Fisher) at room temperature for 1 h. After three washes with Triton X-100, the brain sections were mounted and coverslipped in DAPI Fluoromount-G (SouthernBiotech; Birmingham, AL, USA). Images were captured by Zeiss LSM700 Meta confocal laser microscope (Carl Zeiss). The analysis of mitochondrial segments was performed using ImageJ software, as described previously [16]. In brief, the images of Tom 20 were thresholded, filtered (median, 2.0 pixels), and binarized, followed by separation of continuous structures (size > #x003E;> #x200A;2μm), small particles (size < #x003C;< #x200A;1.5μm), and total particles. The total mitochondrial segments and small mitochondrial segments were normalized as the total mitochondrial area. Continuous structures were analyzed using the percentage of the large particle area to the total mitochondrial area. The 3D cellular reconstruction of Iba-1 images was performed following the description in our previous study [30]. Briefly, the confocal images of Iba-1 were captured as z-stacks by Zeiss LSM700 Meta confocal. The images of Iba-1 were reconstructed using Imaris software (Bitplane AG, Zürich, Switzerland) with 0.4μm smoothing for all channels and images.

Quantification of total ATP content and cytochrome c oxidase activity

The ATP content and cytochrome c oxidase (CCO) activity were determined using a kit of ENLITEN® rLuciferase/Luciferin Reagent (FF2021; Promega, Madison, WI, USA) and an activity assay kit (ab109911; Abcam) according to our previous study [16]. For the measurement of ATP content, the protein samples (30μg) were mixed with the working buffer provided in the assay kit. The values were measured by a standard microplate luminometer (Applied Biosystems, Waltham, MA, USA). The measurement of CCO activity was performed following the manufacturer’s protocol and measured using a Benchmark Plus microplate spectrophotometer (Bio-Rad, Hercules, CA, USA) at 550 nm absorbance.

Inflammatory cytokines assay

The measurement of inflammatory cytokines was performed using Indirect Enzyme-Linked-Immunosorbent-Assay (ELISA) as our previously described [1]. Briefly, the same amount of proteins were loaded into the polyvinyl chloride ELISA microplate (Corning) followed by incubation at 4°C overnight. After washes and incubation with blocking buffer for 1 h, the following antibodies were added: NFκB, TNF-α, IL-1β, IL-4, and IL-10. Thereafter, the plate wells were washed 3 times and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Afterwards, TMB (3, 3’, 5, 5’-tetramethylbenzidine) solution (Thermo Fisher) for 30 min were added and incubated for 30 min following 3 washes. A spectrophotometer (Bio-Rad; Hercules, CA, USA) was used to measure the absorbance at 450 nm. All data were calculated and expressed as changes in optical density value compared to the WT group.

Total antioxidant capacity

The total antioxidant capacity was determined using an antioxidant assay kit (Cayman Chemical, Ann Arbor, MI, USA) following our previously published procedures [2]. Briefly, protein samples were mixed with metmyoglobin and chromogen provided in the assay kit. Thereafter, the working solution hydrogen peroxide solution was added to the plate and incubated for 5 min on a shaker. A spectrophotometer (Bio-Rad; Hercules, CA, USA) was used to measure the absorbance at 450 nm at 750 nm. A Trolox standard curve was used to determine the antioxidant capacity of each sample, and the total antioxidant capacity was calculated and expressed as a percentage change compared to the WT group.

Lipid peroxidation (MDA) assay

Malondialdehyde levels were determined using A lipid peroxidation (MDA) assay kit (ab118970; Abcam, Cambridge, United Kingdom) following the vendor’s instructions. Briefly, the samples were mixed with TBA reagent provided in the kit for 60 min at 95°C. After cooling, the supernatant was moved into the microplate, followed by measurement using a microplate reader at 532 nm. Data were expressed as a percentage change compared to the WT group.

Protein carbonylation determination

An OxiselectTM protein carbonyl ELISA Kit (Cell Biolabs Inc, San Diego, CA, USA) was used to measure protein carbonylation, following the manufacturer’s protocol. In brief, protein samples were loaded onto the protein binding plate provided in the kit and incubated overnight at 4°C. After 3 washes, the samples on the plate were mixed with the dinitrophenylhydrazine (DNPH) working solution and incubated for 45 min at room temperature. After washes, a blocking buffer was added and incubated for 2 h on a shaker at room temperature. After washing, anti-DNP antibodies were added to the plate and incubated for 1 h on a shaker, followed by incubation with an HRP-conjugated secondary antibody for 1 h. Thereafter, the substrate solution was added and incubated for 10 min at room temperature. A microplate reader was used to measure the absorbance of each sample. The values of each sample were calculated using a protein carbonylation ELISA standard curve and expressed as a percentage of the WT group.

Statistical analysis

SigmaStat (Systat Software; San Jose, CA, USA) was used to perform statistical analyses. One-way analysis of variance (ANOVA) followed by Student-Newman-Keuls (S-N-K) post hoc tests was performed to analyze all dependent variables. Multiple group comparisons were conducted with mixed one-way ANOVA and two-way ANOVA to analyze the escape latency in the Barnes maze training trials to determine differences among different groups. A mean±SE was calculated from the data and expressed in all groups (See Supplementary Material). Probability values of p < #x003C;< #x200A;0.05 were considered statistically significant between groups (^*p < 0.05 versus WT group, ^#p < 0.05 versus AD group).

RESULTS

No spatial memory deficits were detected in TgF344-AD rats at 10 months of age

To investigate the spatial memory of 10-month-old TgF344-AD rats, the Barnes maze test was performed at 10 months of age. As shown in training trials (Fig. 2A), there were no significant differences among the three groups in escape latency. Furthermore, time spent in the target quadrant and exploring errors were recorded during the probe trials. As shown in Fig. 2B, no significant differences were observed in quadrant occupancy and exploring errors. The Barnes maze results indicate that AD rats at 10 months of age did not show spatial memory deficits compared to WT animals and AD animals with PBM treatment.

Fig. 2

No spatial memory deficits were detected in TgF344-AD rats at 10 months of age. The Barnes maze task was performed to determine spatial learning and memory. A) Representative tracking plots of rats, escape latency, and escape velocity on the training days were recorded and analyzed. B) Representative tracking plots of rats, escape latency, and quadrant occupancy on the probe trial day were recorded and statistically analyzed. N.S. indicates there are no significant differences among the three groups on the same time point.

PBM alleviated anxious-depressive-like behavior of TgF344-AD rats

We next examined anxious-depressive-like behavior using sucrose preference test, forced swimming test, elevated plus maze test, tail suspension test, and open field test. As shown in Fig. 3A, AD rats exhibited significantly decreased sucrose preference compared with WT rats and PBM-treated animals, suggesting AD rats displayed depressive-like behaviors, which was significantly alleviated by PBM treatment. No significant difference was detected during baseline tests. Furthermore, the forced swim test results confirmed this observation, as evidenced by heightened total immobility time in AD rats and attenuation in PBM-treated animals (Fig. 3B). In addition, the elevated plus maze was performed to test anxiety-like behavior. As shown in Fig. 3C, AD rats performed fewer open arms entries and spent less time in open arms compared with WT animals. However, the open arm entries and time spent in the open arms were significantly increased by PBM treatment, suggesting AD rats presented anxiety-like behavior that was strikingly alleviated in the PBM group. Additionally, as shown in Fig. 3D, AD animals exhibited a significantly elevated immobile duration compared with WT rats. This was attenuated by PBM treatment. We also tested anxiety-related behavior using the open field test. As shown in Fig. 3E, time spent in the center zone, entries into the center zone, and the defecations during testing were recorded and analyzed. As shown in the figure, AD animals presented significantly reduced entries and time spent in the center area and increased defecations in the maze compared with WT animals. PBM treatment prevented these changes in AD rats, further confirming the effect of PBM treatment in alleviating anxiety-related behavior.

Fig. 3

PBM alleviated anxious-depressive like behavior of TgF344-AD rats. Results of the sucrose preference test (A), force swim test (B), elevated plus maze (C), Tail suspension test (D), and open-field test (E). All data are presented as mean±SEM (n = 8–12). *p < #x003C;< #x200A;0.05 versus WT group, ^#p < #x003C;< #x200A;0.05 versus AD group. N.S. indicates there are no significant differences among the three groups on the same time point.

PBM attenuated neuronal apoptosis, neuronal degeneration, and neuronal damage

We next examined the effect of PBM on neurons in the brain. As shown in Fig. 4A, confocal microscopy revealed a significantly amplified number of TUNEL-positive cells in the cortex of AD animals compared with WT rats. Intriguingly, TUNEL-positive cells in AD animals were significantly reduced after PBM treatment, indicating PBM treatment attenuated neuronal apoptosis in the AD brain. To confirm this result, the activity of caspase 3 and caspase 9, essential elements in regulating neuronal apoptosis, were examined using protein samples. As shown in Fig. 4B and 4C, caspase 3 and caspase 9 activities were significantly upregulated in AD groups compared with WT group. However, PBM treatment significantly alleviated these increases. In addition, typical Fluoro-Jade C staining in Fig. 4D showed increased neuronal degeneration in the cortex of AD rats, as evidenced by the elevated number of Fluoro-Jade C positive cells. In contrast, the number of Fluoro-Jade C positive cells was significantly reduced in the PBM-treated group. To examine the effect of PBM on neuronal damage, staining for MBP, MAP2, and PSD 95 was performed. As shown in Fig. 4E-G, the intensity of these neuronal damage markers was significantly decreased in the AD group compared with the WT group, which was preserved by PBM treatment, suggesting PBM treatment can attenuate neuronal damage in AD rats.

Fig. 4

PBM attenuated neuronal apoptosis, neuronal degeneration, and neuronal damage. Results of TUNEL staining (A), caspase 3 (B) and caspase 9 (C) activity, F-jade c staining (neuronal degeneration, D), MBP staining (neuronal damage marker, E), MAP2 (neuronal damage marker, F) staining and PSD95 (postsynaptic density protein, G) in the cortex. Scale bar = 10μm. All data are presented as mean±SEM (n = 6–8). *p < #x003C;< #x200A;0.05 versus WT group, ^#p < #x003C;< #x200A;0.05 versus AD group.

PBM inhibited changes of mitochondrial dynamics and mitochondrial function in TgF344 rats

Mitochondrial dysfunction occurs in a variety of neurodegenerative diseases. Therefore, we next investigated mitochondrial dynamics and mitochondrial function. As shown in Fig. 5A, both mitochondrial total and small particles were significantly increased in AD groups compared with the WT group. By contrast, continuous structures were significantly decreased in AD groups. Intriguingly, these changes were dramatically attenuated by PBM treatment in AD animals, suggesting that mitochondrial fragmentation was significantly ameliorated by PBM treatment. In addition, as shown in Fig. 5B and 5C, CCO activity and ATP production were significantly decreased in AD group rats compared with those in the WT group. In contrast, CCO activity and ATP content were significantly elevated in the PBM group compared to AD animals, suggesting that PBM effectively improves mitochondrial function by increasing CCO activity and improving ATP production. Taken together, this evidence supports PBM inhibited the changes of mitochondrial dynamics and mitochondrial function.

Fig. 5

PBM inhibited changes in mitochondrial dynamics and mitochondrial function in TgF344 rats. A) Representative confocal microscopy images of Tom 20 staining from the cortex. Acquired images of Tom20 staining were processed using ImageJ software to analyze total particles, small particles (size < #x003C;< #x200A;1.5μm), and continuous structures (size > #x003E;> #x200A;2μm). B) Cytochrome c oxidase activity and (C) ATP levels were measured. Scale bar = 10μm. All data are presented as mean±SEM (n = 6–8). *p < #x003C;< #x200A;0.05 versus WT group, ^#p < #x003C;< #x200A;0.05 versus AD group.

PBM suppressed neuroinflammation and oxidative stress

Neuroinflammation and oxidative stress are essential pathological features of AD and contribute to mitochondrial dysfunction and disorder progression. As shown in Fig. 6A, representative confocal microscopy of Iba1 shows upregulated Iba-1 expression compared with WT groups, consistent with other studies. PBM treatment, however, was able to significantly alleviate this change. Further analysis using Image J software confirmed the over-activation of microglia in AD animals, as evidenced by enhanced Iba-1 intensity and microglial cell body diameter in AD groups, whereas these changes were significantly suppressed by PBM treatment. To further confirm the effect of PBM treatment on neuroinflammation, pro-/anti-inflammatory cytokines in the cortex were analyzed by ELISA assays. In line with the results of Iba-1 staining, changes of inflammatory cytokines in AD rats were alleviated by PBM treatment, suggesting that PBM can suppress neuroinflammation in AD (Fig. 6B).

Fig. 6

PBM suppressed neuroinflammation at the early stages of AD. A) Representative confocal microscopy images from the cortex of Iba-1 and 3D rendered images. The intensity of Iba-1 and the diameter of microglial cell bodies were analyzed. B) Pro-inflammatory cytokines NFκB, TNF-α, IL-1β, and anti-inflammatory cytokines IL-4 and IL-10 were measured. Scale bar = 10μm. All data are presented as mean±SEM (n = 6–8). *p < #x003C;< #x200A;0.05 versus WT group, ^#p < #x003C;< #x200A;0.05 versus AD group.

Oxidative stress is closely related to mitochondrial dysfunction and neuroinflammation. Therefore, we next investigated the effects of PBM treatment on oxidative stress. Total antioxidant capacity was measured using cortex protein, and as shown in Fig. 7A, total antioxidant capacity was significantly compromised in AD rats compared with the WT group. However, the decreased total antioxidant capacity in AD animals was significantly abated with PBM treatment. In addition, SOD2, a mitochondrial antioxidant enzyme, was measured by immunofluorescence staining. As shown in Fig. 7B, SOD2 intensity was significantly decreased in AD animals than WT rats, which was preserved by PBM treatment. Furthermore, the products of oxidative stress were measured in Fig, 7C-F. Lipid peroxidation was measured by a lipid peroxidation assay kit using cortex proteins and immunostaining using brain slices. In AD animals, lipid peroxidation was significantly increased compared with WT rats, which was significantly suppressed by PBM treatment (Fig. 7C-D). Furthermore, protein carbonyls and DNA double-strand breaks (p-H2A.X Ser139) were measured and illustrated in Fig. 7E-F. As shown in Fig. 7E-F, both the levels of protein carbonyls and the intensity of p-H2A.X were significantly increased in AD rats. However, PBM treatment inhibited these changes, indicating PBM treatment could suppress the elevated oxidative damage to DNA and proteins in AD animals.

Fig. 7

PBM suppressed oxidative stress at the early stages of AD. Total antioxidant capacity (A), SOD2 (B), MDA levels (C), 4-HNE expression (D), protein carbonyl levels (E), and p-H2A.X staining (DNA double-strand breaks) (F) in the cortex were measured. The results of the AD group and the PBM group are quantified as percentage changes versus the WT group. Scale bar = 10μm. All data are presented as mean±SEM (n = 6–10). *p < #x003C;< #x200A;0.05 versus WT group, ^#p < #x003C;< #x200A;0.05 versus AD group.

DISCUSSION

The present study provides evidence that PBM treatment protected against anxious-depressive-like behavior in the early stages of AD pathogenesis in a novel AD rat model when initiated at 2 months of age. Our results demonstrate that PBM treatment conferred protective effects on reducing neuronal apoptosis, neuronal degeneration, and neuronal damage. Serving as a non-invasive treatment, the beneficial effects of PBM treatment can be attributed to, in part, its ability to improve mitochondrial function. Mitochondrial fragmentation, present in clinical AD and experimental models, was effectively attenuated by PBM treatment. These improvements in mitochondrial function and the maintenance of mitochondrial structure alleviated oxidative stress and neuroinflammation. Thus, these findings suggest that PBM has potential as a treatment method to improve anxiety and depression in AD patients at the early stages of AD and may be a promising option to delay AD progression.

Memory loss is a typical AD symptom and is often the first impetus for patients to seek diagnosis [31, 32]. However, brains of AD patients exhibit amyloid plaque accumulation, hyperphosphorylated tau, and neuronal loss before clinically diagnosable learning and memory impairment [2, 5]. In the present study, we found that AD animals at 10 months of age did not show learning and memory deficits, consistent with previous studies [2, 24]. However, anxiety and depression have been recognized as early endophenotypes in AD, occurring before learning and memory decline [2 , 33–35]. As one of the most prevalent mental illnesses, depression is highly prevalent in patients with severe cognitive deficits [36]. Studies suggested that depression is a risk factor for AD, and some of the pathophysiological mechanisms have overlap between AD and depression [36]. Patients with depression or depressive symptoms present a greater than 2-fold risk of dementia [37]. Furthermore, according to previous studies, hippocampal plaque and neurofibrillary tangles were formed in some patients with a lifetime history of depression [38, 39]. In addition to depression, anxiety is often identified in patients with AD [40].

In this AD rat model, anxiety-like behavior is considered as an early behavioral phenotype [5]. The delay of AD pathology was also accompanied by decreased anxiety-like behavior [24]. In the current study, we found PBM was capable of alleviating anxious-depressive-like behavior in AD rats, as evidenced by numerous behavioral tests that were widely used in the measurement of anxiety and depression. Consistent with the improvements in anxious-depressive-like behavior, we confirmed the inhibitory effect of PBM treatment on neuronal apoptosis, neuronal degeneration, and neuronal damage, which along with DNA fragmentation, have been found in patients with depression and anxiety [41, 42]. Our findings were in line with previous human trials and animal models of PBM for depression and anxiety [23 , 43–45]. Cassano et al. found PBM treatment improved depressive and anxious symptoms in patients suffering from major depressive disorder and generalized anxiety disorder [43 –45]. The beneficial effects of PBM treatment on depression were also reported by Henderson et al. in a human study [46]. Furthermore, in a depressive mouse model, 30-min daily PBM treatments (23 mW/cm²) for 28 days significantly attenuated depressive-like behaviors, which is in line with our current study [23]. Therefore, PBM treatment may be a potential approach to reduce anxious-depressive-like behavior in AD patients.

In our study, PBM was directly delivered to the scalp without causing a significant cortical temperature increase using a continuous-wave 808 nm laser [17, 47]. Complex IV (cytochrome c oxidase, CCO) in mitochondria is considered the primary target of PBM [8 , 49]. In brain disorders including AD, depression, and anxiety, diminished CCO activity contributes to and perpetuates mitochondrial dysfunction [49 –51]. In the present study, we found that decreased CCO activity in AD can be significantly restored by PBM treatment. This was further supported by increased ATP production in the PBM group. These results suggest that the beneficial effect of PBM is, at least in part, due to accelerated CCO activity. Furthermore, mitochondrial fragmentation emerges at the early stage of AD pathogenesis and induces learning impairments [52, 53]. In line with this evidence, our study demonstrated that mitochondrial fragmentation was highly elevated in AD rats at 10 months of age, and notably, PBM treatment effectively attenuated these elevations. The mitochondria are the primary source of reactive oxygen species (ROS), so these findings regarding the role of PBM treatment in regulating mitochondrial function and fragmentation suggest that oxidative stress may contribute to the improvement of anxious-depressive-like behavior in this transgenic AD rat model. In our study, PBM was directly applied to the brain with an irradiance of 350 mW/cm² for the scalp and 25 mW/cm² for the cortex. However, because our study was performed on an animal model, we still do not know how much of each PBM dose will be lost if applied to the human brain. Notably, several important studies demonstrated that the beneficial effects of PBM are limited to the irradiated area and have indirect effects on distant, non-irradiated tissues or organs [54 –58]. Gordon et al. defined this phenomenon as “remote PBM” [54]. For the remote PBM, PBM treatment is applied on peripheral tissue and confers indirect or body-wide beneficial effects, which is helpful for addressing the issues of light penetration across the human scalp and skull [54]. Therefore, future studies should determine whether remote PBM or transcranial PBM at 25 mW/cm² for the cortex in the human brain has similar effects in the improvement of anxious-depressive-like behavior at the early stages of AD.

Oxidative stress depends on the balance of oxidation-reduction reactions, wherein the body’s antioxidant defense system competes with the burden of ROS generation [59]. Previous studies detected a close relationship between neurodegenerative diseases and mental neuropsychiatric disorders [59, 60]. In patients with anxiety disorders, measures of oxidative stress reveal a high level of oxidative damage alongside a compromised antioxidant defense system [61, 62]. For patients with depression, there are significantly lower levels of critical antioxidants contributing to a decreased total antioxidant capacity [63]. Higher MDA and protein carbonyl levels are associated with an increased degree of oxidative stress in depression and anxiety [2, 64]. Furthermore, in a a mouse stress model with anxiety-like behaviors, transcranial PBM treatment significantly alleviated oxidative stress and anxiety-like behaviors [65]. In line with these findings, we found AD rats with anxious-depressive-like behavior exhibited impaired total antioxidant capacity and compromised SOD2 expression. However, PBM treatment could alleviate diminished antioxidant capacity and improve the expression of SOD2. To support this finding, our observations also found that PBM could attenuate the oxidative damage to proteins, lipids, and DNA, as evidenced by decreased protein carbonyls, MDA levels, 4-HNE, and p.H2A.X expression in the PBM group. Taken together, our results demonstrate that PBM treatment is capable of maintaining the antioxidant capacity and reducing oxidative damage to critical biomolecules.

Neuroinflammation is another crucial factor that contributes to neuronal damage in brain disorders [66]. Microglial overactivation is consider as an early pathogenic event that contributes to neuropil destruction in AD patients [67]. Pro-inflammatory cytokines such as NFκB, TNF-α, and IL-1β released from microglia are present in AD pathology and confer neurotoxic effects on neurons [68, 69]. The overactivation of microglia and amplified pro-inflammatory factor release have also been found in patients with depression [70]. The level of IL-1β was elevated in depressed elderly patients and closely related to the severity of depression [71]. Similarly, higher pro-inflammatory cytokine levels were also associated with more severe anxiety symptoms [72 –74]. In a previous study using a mouse model of restraint stress, PBM combined with coenzyme Q10 treatment significantly reduced chronic restraint stress-induced neuroinflammation in the prefrontal cortex and hippocampus [75]. Consistent with previous findings, our observations showed that AD animals with anxious-depressive-like behavior presented microglial overactivation, and a shift toward increased pro-inflammatory cytokine release. Intriguingly, PBM treatment alleviated these changes and improved the levels of anti-inflammatory cytokines. Taken together, our results demonstrate that PBM can inhibit microglial over-activation and alleviate neuroinflammation in AD rats.

In conclusion, using an AD rat model with anxious-depressive-like behavior, our study demonstrates that PBM treatment can alleviate anxious-depressive-like behavior, as well as neuronal loss and neuronal damage. The preservation of mitochondrial function and integrity and the alleviation of neuroinflammation, excessive microgliosis, and oxidative stress after PBM treatment may contribute to the beneficial effects of PBM on anxious-depressive-like behavior at the early stages of AD development. Although more work is still needed to investigate the effect of PBM on AD pathology and AD progression, the results of our study demonstrate that, PBM treatment could benefit AD patients suffering from depression and anxiety.

Footnotes

ACKNOWLEDGMENTS

This study was supported by research grants from the United States of America: NS086929 from the National Institute of Neurological Disorders and Stroke; AG058603 from the National Institute of Aging, National Institutes of Health.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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