Nano-PSO Administration Attenuates Cognitive And Neuronal Deficits Resulting From Traumatic Brain Injury Part 1

Abstract: Traumatic Brain Injury (TBI), is one of the most common causes of neurological damage in young populations. It is widely considered a risk factor for neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's (PD) disease. 

Traumatic brain injury is a worrying condition that can cause serious damage to our brains, affecting us to varying degrees in memory, thinking, and decision-making. However, although these problems may affect our lives for some time, there are still many positive measures that can help us recover or improve our memory.

First, we can help improve our memory through a series of cognitive training, which can include playing games, completing daily tasks, and stimulating our brains by learning new things. This training can help us develop new neural pathways, improve our thinking ability, and enhance our memory.

Second, in daily life, we can help us maintain a healthy brain by changing our diet and exercise habits. Some foods rich in protein, vitamins, and antioxidants, such as vegetables, fruits, and nuts, can help us improve our cognitive abilities and protect our brains from damage. In addition, regular exercise and maintaining good sleep quality can also be beneficial to brain recovery and health care.

Finally, we can support and guide our recovery process by staying in touch with family, friends, and healthcare professionals. Communicating with others and receiving help and support can help us maintain an optimistic attitude, which is essential for our brain recovery and memory improvement.

In conclusion, although traumatic brain injury may bring some challenges, we can overcome these difficulties and improve our memory and cognitive abilities through positive actions. Remember, everyone can achieve positive results through hard work and patient efforts. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains adequate nutrition and energy, thereby improving brain vitality and endurance.

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These diseases are characterized in part by the accumulation of disease-specific misfolded proteins and share common pathological features, such as neuronal death, as well as inflammatory and oxidative damage. 

Nano formulation of Pomegranate seed oil [Nano-PSO (Granagard TM)] has been shown to target its active ingredient to the brain and thereafter inhibit memory decline and neuronal death in mice models of AD and genetic Creutzfeldt Jacob disease. 

In this study, we show that administration of Nano-PSO to mice before or after TBI application prevents cognitive and behavioral decline. In addition, immunohistochemical staining of the brain indicates that preventive Nano-PSO treatment significantly decreased neuronal death, reduced gliosis, and prevented mitochondrial damage in the affected cells. 

Finally, we examined levels of Sirtuin1 (SIRT1) and Synaptophysin (SYP) in the cortex using Western blotting. 

NanoPSO consumption led to higher levels of SIRT1 and SYP protein postinjury. Taken together, our results indicate that Nano-PSO, as a natural brain-targeted antioxidant, can prevent part of TBI-induced damage.

Keywords: TBI; Nano-PSO; neuroinflammation; oxidative stress; neurodegeneration

1. Introduction

Traumatic Brain Injury (TBI) is a profound public health concern and a major cause of disability and death in young adults and elderly populations. It is considered one of the most common neurological injuries that causes morbidity and mortality and affects more than 10 million people worldwide each year [1]. Since head injuries do not always require seeking proper medical care, these data are likely even higher. 

The main causes of TBI include falls, road accidents, assaults, and sports injuries [2]. TBI is common among men and especially frequent in adolescents and young adults following road accidents and alcohol-related injuries. 

It is also common in the elderly due to their increased chance of falls [3]. People who have had TBI may suffer from behavioral, cognitive, and emotional impairments, both short-term and long-term, depending on the severity of the injury [3]. 

Patients exposed to TBI can emerge unscathed but can also be exposed to injuries that require prolonged hospitalization and even end in death. About 40% of cases of severe injury post-TBI will end in death, with survivors suffering from various neurological disorders including epilepsy, dementia, and neurodegenerative diseases [3,4]. 

Moderate injury causes morphological changes similar to those of severe injury, but they are less severe, and the cognitive damages are mostly milder than in severe injury [5]. In addition, there is well-established evidence that TBI is an important risk factor for the development of neurodegenerative diseases such as Alzheimer's and Parkinson's [6–8]. 

Oxidative stress is one of TBI's potential secondary injuries, which also includes excitotoxicity, inflammation, apoptosis, and mitochondrial dysfunction. Mitochondrial dysfunction is often found in patients with neurodegenerative diseases such as Parkinson's and Alzheimer's [9–12]. 

Mitochondrial dysfunction can lead to the unregulated overproduction of reactive oxygen species (ROS), which is a frequent indicator of TBI secondary injury [13]. The human body produces endogenous antioxidants to offset or inhibit potential increases in ROS production. 

When TBI leads to an increase in ROS production, the body's natural antioxidant levels are overwhelmed often causing lipid peroxidation and DNA and protein damage [14]. This can lead to either an overage of ROS or low antioxidant levels, a condition known as oxidative stress that can have devastating and often toxic effects, causing the death of neuronal cells and potential pathogenesis [15–19]. 

TBI has no proven treatment for lessening damage or even prevention. For this reason, it is of paramount importance to seek a better understanding of the pathological mechanisms that follow injury to determine ideal treatment protocols. 

Nano-PSO (Granagard TM), a nano-formulation of pomegranate seed oil comprising high levels of punicic acid, the strongest natural antioxidant, was shown to ameliorate neurodegeneration signs, including memory and cell death in several models of neurodegenerative diseases [20–22]. 

In the transgenic mice model for genetic CJD, long-term administration of Nano-PSO resulted in a delay in disease progression and reduced cell death. In the 5XFAD mice model for AD, Nano-PSO administration prevented memory impairment and reduced the accumulation of A-beta, both intracellularly and intracellularly. 

In all models, Nano-PSO prevented a reduction in mitochondrial activity, probably because of its strong and brain-targeted antioxidant function. In humans, Nano-PSO was recently shown to increase memory in multiple sclerosis patients [23]. 

In the present study, we show that the administration of Nano-PSO, a dietary supplement both before or after TBI in mice, mitigates the impairments of visual and spatial memory. 

It also reduces neuronal loss in the temporal cortex and dentate gyrus and reduces reactive astrocyte intensity in the temporal cortex of injured mice brains. Moreover, it restores the production of cytochrome C oxidase (COX), a mitochondrial enzyme that is reported to be affected in brain cells during neurodegeneration [24]. 

Furthermore, it plays a role in the respiratory chain and is responsible for the reduction of up to 95% of the oxygen taken up by the cells, including neural cells [24].

2. Results

2.1. Administration of Nano-PSO to Mice before and after TBI Prevents Their Cognitive Deterioration

Following TBI exposure and treatment with Nano-PSO either as a preventive or as a post-TBI treatment (see Figure 1 and methods for scheme of the experiments), male ICR mice were evaluated using the elevated plus maze at two weeks post-TBI among separate cohorts. 

We evaluated the anxiety-like behavior of the mice by recording the time spent in the open arms of the maze and the total number of entrances to each arm, respectively. 

There were no differences in the time spent in the open arms between all groups at both treatment approaches tested, indicating that the anxiety-like behavior of the mice was not affected either by the injury or by treatment with Nano-PSO (results not shown). 

To assess the effects of both of the treatment approaches (pre- or post-TBI) of NanoPSO consumption on memory formation, the novel object recognition (NOR) and Y-maze paradigms were performed on different groups of mice at two weeks post-injury. 

The NOR paradigm is consistently reported to be impaired by TBI [25]. As expected, TBI mice suffered from significant visual memory deficits and spent less time near the novel object, compared to all the other groups (p < 0.01, p < 0.001, Figure 2A, B). 

The deficit was ameliorated by Nano-PSO in both of the treatment approaches. Next, we performed the Y-maze test to evaluate the spatial memory as previously described [26]. 

At both treatment approaches, TBI mice experienced spatial memory impairment compared to untreated control mice (p < 0.001, Figure 3A, B). When mice consumed the treatment by Nano-PSO, spatial memory impairment was ameliorated in both treatment approaches (p < 0.001).

Figure 3. Nano-PSO administration to TBI mice improved spatial memory assessed in the Y-maze test. TBI mice treated or untreated were subjected to the Y maze test at two weeks following TBI as described in the methods: (A) mice treated with Nano-PSO before TBI; and (B) mice treated with Nano-PSO post-TBI represent preference index of the relative time that mice spent exploring the novel arm compared to the old arm. 

Statistical analysis by One-way ANOVA revealed that TBI animals had a deficit in spatial memory compared to all other groups. (A: F (3, 36) = 16.039, p = 0.000 Fisher's LSD post hoc, *** p < 0.001, n = 10; B: F (1, 15) = 14.127, p = 0.00 Fisher's LSD post hoc, ** p < 0.01, n = 10). NS = Not significant. Values are presented as mean ± SEM.

2.2. Nano-PSO Treatment Prevents Neuronal Death in TBI Mice

Following the behavioral experiments, which found that Nano-PSO can prevent cognitive damage resulting from brain injury, we looked into the levels of TBI brain damage in the presence or absence of Nano-PSO on cellular levels by immunocytochemistry. 

Mice were terminated and their brains were harvested three weeks post-injury, at which time further biochemical changes were found [25,27]. 

The changes were examined in the temporal cortex, CA3 region of the hippocampus, and dentate gyrus on both sides of the brain. The results from both sides of the brain were combined into one value for each mouse. 

Figures 4A and B show the immunostaining of these brain areas with anti-NeuN antibody, a marker of mature neurons. The results show that the number of neurons was significantly reduced following TBI, but such reduction in neurons was prevented if Nano-PSO was administrated before TBI or post-TBI and until mice were terminated (three weeks later). 

TBI induced a decline in the neuronal counts within the cortex and dentate gyrus compared to all other groups (** p < 0.01, *** p < 0.001), indicating a significant neuronal loss at three weeks following the injury (Figure 4A, B). Both of the treatment approaches preserved neuronal counts in the cortex and dentate gyrus. 

However, preventive treatment further mitigated the neuronal loss in these areas (** p < 0.01, *** p < 0.001) (Figure 4A) compared to the treatment after TBI (* p < 0.05) (Figure 4B).

2.3. Administration of Nano-PSO Reduces Inflammation Caused by TBI

Evaluation of neuro-inflammatory processes occurring after TBI exposure was obtained by immune-histochemical staining using GFAP antibody which labels reactive astrocytes [28,29]. 

The staining was performed to examine whether there were changes in the expression level of the astrocytes TBI three weeks after injury and whether a preventive or post-TBI treatment with Nano-PSO affected these changes. Brain slices obtained from the temporal cortex were studied three weeks postinjury. 

As can be seen in Figure 5, TBI exposure induced a significant elevation in astrocytes in the cortex (p < 0.001, p < 0.01), respectively (Figure 5). When Nano-PSO was administrated as a preventive or as a treatment post-TBI, it led to a significant reduction in the activated astrocytes in the cortex, p < 0.001, p < 0.05, respectively (Figure 5). 

These results demonstrate that due to TBI, as from untreated TBI, treated with Nano-PSO before TBI and treated with Nano-PSO post-TBI were immunostained for NeuN (green) and DAPI (blue) [scale bar 50 µm]; (B) quantitative assessment of NeuN immunostaining for all sections (bregma temporal cortex −1.06 nm and dentate gyrus −1.34 nm) at the end point of each experiment was quantified using One-way ANOVA (Cortex: PRE (F (3, 16) = 15.469, p = 0.000 Fisher's LSD post hoc, *** p < 0.001, N = 5); PSOT (F (3, 12) = 8.607, p = 0.000 Fisher's LSD post hoc, * p < 0.05 ** p < 0.01, n = 3–5)), (DG: PRE (F (3, 14) = 6.804, p = 0.000 Fisher's LSD post hoc, ** p < 0.01, n = 4–5); PSOT (F (3, 13) = 12.580, p = 0.000 Fisher's LSD post hoc, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3–4)). NS = Not significant. Values are presented as mean ± SEM.

2.4. Nano-PSO Restores COX IV1 Activity following TB1

It was shown previously that in spontaneous/genetic models of neurodegenerative diseases, such as AD and CJD, the expression of COX IV1, an important mitochondrial enzyme for energy production in cells, is drastically reduced and replaced by COX IV2, which can function under high levels of ROS [24,30]. However, this is a temporary effect until such levels of ROS finally result in apoptosis and cell death. 

Nano-PSO administration reduces ROS levels and restores COX expression [30]. Therefore, in this work, we tested if this is also the case for TBI, an outside injury caused by normal mice. 

To this effect, we immunostained brain slices as described above with COX IV1 antibody. Figure 6 shows that, as was the case for genetic diseases, TBI also causes a reduction in the levels of COX IV1 even three weeks after the infliction of the damage in the brain. 

In this case, and as shown before [30,31], the administration of Nano-PSO increases COX IV1 levels both in the control and in the pre-and post-TBI mice. Since an increase in COX IV1 levels reflects the oxidative status of cells, we conclude that Nano-PSO can also reduce ROS levels in control mice and not only in the TBI-affected mice.

Figure 6. Nano-PSO treatment restored mitochondrial COX IV1 expression in all mice: (A) Coronal sections through the temporal cortex of brains from treated and untreated controls, as well as from untreated TBI, treated with Nano-PSO before TBI and treated with Nano-PSO post-TBI were immunostained for COX IV1 (red) and DAPI (blue) [scale bar 50 µm]; (B) Quantitative assessment of COX IV1 immunostaining for all sections (bregma temporal cortex −1.06 nm) at the endpoint of each experiment was quantified by One-way ANOVA (F (4, 12) = 45.101, p = 0.000 Fisher's LSD post hoc, ** p < 0.01, *** p < 0.001, n = 3–4). NS = Not significant. Values are presented as mean ± SEM.

2.5. Nano-PSO Elevates SIRT1 and SYP Levels Post-TBI

The Immunoblot of the total levels of SIRT1, a marker for NAD-dependent deacetylase sirtuin-1 in the cortex, was examined for all mice three weeks post-TBI (Figure 7). A reduction of SIRT1 levels was found. 

These results are in line with a previous study with a mild TBI model from our lab [32]. However, this reduction was prevented with Nano-PSO when it was administrated immediately for several weeks after the event. 

Figure 7B shows an immunoblot of total levels of synaptophysin (SYP), a marker for synaptic structures, from brain homogenates of control mice treated or untreated with Nano-PSO as well as from TBI or post-TBI-treated mice. 

The results show that, concomitant with the neuronal death shown in Figures 4A,  and B, synaptic damage was also caused by TBI. However, this damage can be prevented if a strong and brain-directed antioxidant such as Nano-PSO is administrated immediately for several weeks after the infliction of the damage.

It has been shown before that most of the affected neurons did not die immediately after the injury but in the days after due to the extensive oxidative stress caused by TBI [33]. Therefore, immediate antioxidant treatment can be efficient enough to prevent extensive damage in the long run.

Figure 7. The impact of Nano-PSO on SIRT1/SYP expression in the cortex of TBI mice: (A) Brain homogenates from the right cortex of treated and untreated controls, as well as from TBI and NanoPSO-treated post-TBI mice were immunoblotted with SIRT1 and TUB detected at (110 KDa, 50 KDa), respectively. Quantitative analysis of immunoreactive bands of SIRT1 was quantified by Image J and normalized against the control protein TUB. 

Levels of SIRT1 were significantly reduced 30 days postinjury in the cortex of TBI mice compared to the control. Nano-PSO treatment prevented this reduction ** p < 0.01. One-way ANOVA revealed a significant elevation in the expression of SIRT1 in mice that received the treatment postinjury. (F (3, 20) = 1.084, p = 0.002, N = 5 Fisher's LSD post hoc). 

(B) Brain homogenates from the right cortex of treated and untreated controls, as well as from TBI and Nano-PSO treated post-TBI mice were immunoblotted with SYP and TUB detected at (37 KDa, 50 KDa), respectively. 

Quantitative analysis of immunoreactive bands of SYP was quantified by Image J and normalized against the control protein TUB. 

One-way ANOVA revealed a significant elevation in the expression of SYP in mice that received the treatment post-TBI compared to the TBI group (F (3,20) = 1.084, p = 0.379, Fisher's LSD post hoc * p < 0.5, n = 4–5). NS = Not significant. Values are presented as mean ± SEM.

Below each graph, a representative image of the levels of SIRT1/SYP and the household protein tubulin (TUB) in the right cortex among all the groups is presented.

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