Synergistic Action of Vitamin D3 and A Protects Motor Activity by Regulating Reactive Astrocytes, Inflammatory Cytokines, and Dopaminergic Activity in the Corticobasal Loop of a Mice Model of Extrapyramidal Syndrome

doi:10.21203/rs.3.rs-3749691/v1

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Research Article

Synergistic Action of Vitamin D3 and A Protects Motor Activity by Regulating Reactive Astrocytes, Inflammatory Cytokines, and Dopaminergic Activity in the Corticobasal Loop of a Mice Model of Extrapyramidal Syndrome

https://doi.org/10.21203/rs.3.rs-3749691/v1

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Background:

Extrapyramidal syndromes (EPS) represent neurological side effects of antipsychotic medications, characterized by motor disturbances. While previous studies have indicated the neuroprotective effects of vitamin D and A against EPS, the underlying mechanisms of this protection remain unclear.

Methods:

Twenty-four adult male mice were categorized into four groups: positive and negative control groups, one receiving a dopamine antagonist, and the other receiving both a dopamine antagonist and vitamins D and A. Sections of the corticobasal loop, specifically the motor cortex (M1) and basal nuclei (CPu), were prepared for Immunohistochemistry (IHC) and stained with Glial Fibrillary Acidic Protein (GFAP) to visualize active astrocytes. ELISA assays for TNF-α, IL-6, IL-4, IL-13, and dopamine levels were performed on homogenized brain sections.

Results:

The EPS group exhibited a significant increase in TNF-α and IL-6 levels in M1 and CPu. Treatment with dopamine agonists and vitamin D/A resulted in significant reductions in IL-6 levels. Only the Vitamin D/A group showed a significant decline in TNF-α. The EPS group recorded significant decreases in IL-4 and IL-13, with IL-13 significantly elevated in the dopamine agonist and Vitamin D/A groups. IL-4 was notably increased in the Vitamin D/A groups. Dopamine concentration significantly declined in the EPS group, with improvements observed in the groups treated with dopamine agonists, vitamin D, and A. Reactive astrocytes were significantly expressed in the M1 and CPu of the EPS group but poorly expressed in other regions.

Conclusions:

EPS is linked to astrocyte activation, an upsurge in pro-inflammatory cytokines, a decline in anti-inflammatory cytokines, and dopamine in the corticobasal loop. Administration of vitamin D3 and A was found to exert its effects by suppressing pro-inflammatory cytokines and repressing anti-inflammatory cytokines associated with astrocyte activation.

Extrapyramidal syndrome

Corticobasal loop

Inflammation

Activated astrocytes.

Extrapyramidal syndromes (EPS) represent neurological side effects induced by antipsychotic medications, primarily acting on dopamine receptors in the brain [1–5]. These manifestations include movement disorders such as akathisia, dystonia, Parkinsonism, and tardive dyskinesia [5–9]. The dopaminergic system's crucial role in motor function has been extensively documented, with prolonged dopamine receptor inhibition affecting neurons in the primary motor cortex and basal nuclei [10–15].

Haloperidol therapy, a commonly used antipsychotic, has been associated with decreased activation of the corticobasal loop during motor tasks, disrupting immune responses and inducing neuroinflammatory and neurodegenerative changes [16–18]. The corticobasal loop is particularly vulnerable to these changes, contributing to EPS pathogenesis [19–21].

In response to neuroinflammation, astrocytes undergo activation, characterized by morphological, molecular, and functional remodeling [22–25]. Activated astrocytes, identified by glial fibrillary acidic protein (GFAP) immunohistochemical staining, exhibit hypertrophy and proliferation, serving as pathological indicators of central nervous system (CNS) lesions [26, 27].

Tumor necrosis factor alpha (TNF-α) is identified as a key trigger for astrocytic activation, playing a pivotal role in inflammatory responses within the brain. Interleukin (IL)-6, another pro-inflammatory cytokine, contributes to the acute phase of the inflammatory response [28–30]. Conversely, IL-4 and IL-13, categorized as anti-inflammatory cytokines, have been linked to allergic inflammation, and a decrease in their levels is associated with neurodegenerative diseases [32–36].

While previous studies reported motor function loss, increased cytotoxicity, and oxidative stress in preclinical EPS models [37], specific pathophysiological features such as astrocyte activation and cytokine levels remain unexplored. Understanding the role of astrocytes and inflammatory cytokines in response to dopamine inhibition-induced brain tissue damage could unveil pathways influencing neuron preservation or deterioration in EPS, presenting potential treatment targets.

Several studies suggest the neuroprotective effects of supplemental vitamin D3 and A combinations in neurodegenerative diseases. This study aims to assess the neuroprotective impact of vitamin D3 receptor heterodimer activation through the administration of a vitamin D3 and A supplement. The focus is on regulating astrocyte-mediated neuroinflammatory processes and dopamine levels in a mouse model of extrapyramidal syndrome.

This research seeks to elucidate the intricate connections between astrocytic activation, inflammatory cytokines, and dopamine levels in EPS pathogenesis. Additionally, the potential neuroprotective effects of vitamin D3 and A supplementation are explored, offering insights into novel therapeutic avenues for EPS and related disorders.

2.1 Experimental animals

2.1.1 Experimental Design:

A total of twenty-four (24) male mice, with an average weight of 29.0 g ± 2.0, were obtained for this study. The mice were housed in a well-ventilated room maintained at room temperature (27ºC), with a 12-hour light and dark cycle. Adequate care was taken to ensure optimal conditions for the animals' well-being.

2.1.2 Housing and Nutrition:

The mice had ad libitum access to mouse chow and were provided with a constant supply of clean drinking water to meet their nutritional needs. This was crucial in maintaining their health and ensuring consistent hydration throughout the experimental period.

2.1.3 Animal Welfare Protocols:

All protocols for handling and caring for the animals adhered strictly to the guidelines set forth by the National Health Institute (NIH) and the Institutional Animal Care and Use Committee (IACUC). Additionally, ethical approval for the animal care and experimental procedures was obtained from the Animal Care and Use Research Ethics Committee (ACUREC) of Bayero University, Kano, Nigeria.

This commitment to following established guidelines and obtaining ethical approval underscores the study's dedication to responsible and humane treatment of the research subjects, ensuring the integrity and ethical standards of the research process.

2.2 Animal treatment

A model of EPS was developed using 10 mg/kg body weight (BW) of a dopamine-2 receptor blocker, in line with an earlier protocol [15, 37]. 800 IU of vitamin D3 and 100 IU of vitamin A were used for vitamin D3 and vitamin A treatments, respectively. Five (5) mg/kg BW of a dopamine-2 receptor agonist (+ D2) and bromocriptine were used as standard treatment for PD. Details of the animal treatment are given in Table 1.

Table 1. Animal treatment

2.3 Animal Sacrifice

On the last day of the treatment phase, the experimental animals were anesthetized using 50 mg/kg of ketamine. Subsequently, the animals underwent transcardial perfusion through the left ventricle, involving the perfusion of 50 ml of normal saline to flush out blood and 50 ml of 10% formalin to fix the brain. The skull was then opened using forceps to harvest the whole brain, which was further fixed.

2.4 Biochemical Analysis

Some brain tissues that were not perfused were submerged in an isotonic solution of 0.25M sucrose (β-D-fructofuranosyl-α-D-glycopyranoside; C12 H22O11; Mol. Wgt = 342.30) (Sigma). Before biochemical analysis, the sucrose solution was maintained at 4°C. Using a mortar and pestle, an equal weight of tissues from each group was homogenized in the sucrose solution. Subsequently, the mixture was placed in 20-ml polypropylene tubes and centrifuged while balancing the load in a Thermoscientific Multifuge 3SR + with the temperature set at 4°C and the speed at 3,000 rpm for 30 minutes [31]. The supernatants were utilized for determining the concentrations of pro and anti-inflammatory cytokines as well as dopamine in brain tissues.

2.5.1 Determination of Cytokines

ELISA analysis of pro-inflammatory cytokine levels

Determination of TNF-α

TNF-α levels in the motor cortex and striatum were measured using the colorimetric detection method of the Mouse TNF-α ELISA Kit following the manufacturer’s instructions.

Determination of IL-6

TNF-α levels in the motor cortex and striatum were measured using the colorimetric detection method of the Mouse IL-6 ELISA Kit according to the manufacturer’s instructions.

ELISA analysis of Anti-inflammatory cytokines

Determination of IL-4

TNF-α levels in the motor cortex and striatum were measured using the colorimetric detection method of the Mouse IL-4 ELISA Kit according to the manufacturer’s instructions.

Determination of IL-13

TNF-α levels in the motor cortex and striatum were measured using the colorimetric detection method of the Mouse IL-13 ELISA Kit according to the manufacturer’s instructions.

2.5.2 Dopamine concentration

The dopamine concentration in the homogenized motor cortex and striatum was assessed using the mouse dopamine ELISA Kit, following the manufacturer’s instructions.

2.5.3 Immunohistochemistry

After fixation, the brain was dissected on a stereotaxic grid and Allen’s mouse brain atlas to obtain sections of the motor cortex and striatum. The diced brain tissues were then transferred into a freshly prepared cryopreservative solution (10% formalin + 30% sucrose) for 72 hours at 4°C. Following this, the brain tissues were processed to obtain paraffin wax-embedded tissue blocks for Immunohistochemistry (IHC).

The tissue sections were brought to water and subjected to epitope retrieval solution (pH 6) for 30 minutes. Subsequently, the tissue blocks were immersed in 1% hydrogen peroxide for 3 minutes. The sections were then incubated in anti-GFAP for 45 minutes at room temperature. Following this, the sections underwent incubation in mouse HRP for an additional 45 minutes. The samples were stained with DAB for 5 minutes and counterstained with Mayer's hematoxylin for 1 minute. This comprehensive procedure aimed to prepare the tissue sections for subsequent IHC analysis, ensuring accurate visualization of the targeted biomarkers.

2.6 Photomicrography and Cell count:

The regions of the cell obtained were approximately predetermined, and the orientation of the tissues prior to processing was maintained consistently. Serial sectioning was performed, and the third section was selected for the staining used in this study. For statistical analysis, five sections in each group were counted per unit area. A light microscope (LCD Bresser), equipped with BMS MicroscopeB, V, BMS_Pix3, version x64, 4.11.18081.20201205, was utilized to capture images of the stained tissue sections. The magnification was set to 100X on the digital camera attachment of the microscope. Cell counting was conducted using Fiji (Just Image J) software version 2.9.0, released on September 14, 2022, facilitating accurate and efficient analysis of the obtained data.

2.7 Statistical Analysis

The quantitative data were presented as the mean ± standard error of the mean. Statistical analysis was carried out using one-way analysis of variance (ANOVA) with a Tukey post-hoc test to identify any significant differences between the groups. The significance level was set at p < 0.05. All statistical analyses were conducted using GraphPad Prism software, Version 7.0 (GraphPad Software Inc., San Diego, CA, USA).

3.1 ELISA analysis of pro-inflammatory cytokine levels

Tumor Necrosis Factor Alpha (TNF-α) levels in Motor cortex and Basal nuclei

Compared to the control group (125.6 ± 9.469), there was a significant increase in TNF-α across the groups: EPS (294.0 ± 12.49) (***P < 0.001), EPS + D2 (246 ± 16.31) (***P < 0.001), and EPS + VD + VA (196.0 + 24.82) (*P < 0.05). When compared to the EPS group, the EPS + VD + VA group exhibited a significant decrease in TNF-α.

Interleukins-6 (IL-6) levels in Motor cortex and Basal nuclei

Compared to the control group (61.00 ± 10.5), a significant increase (**P < 0.01) in IL-6 levels was observed in the EPS group (142 ± 16.85). There were no significant differences between the control group and the EPS + VD + VA (78 ± 15.94) and EPS + D2 (70 ± 15.49) groups. When compared to the EPS group, there was a significant decrease (*P < 0.05) in IL-6 levels in the EPS + VD + VA and EPS + D2 groups.

3.2 ELISA analysis of Anti-inflammatory cytokine levels

Interleukin-4 (IL-4) levels in Motor cortex and Basal nuclei

The EPS (37.00 ± 5.385) group shows a significant decrease (**P < 0.01) in IL-4 when compared to the control (69.00 ± 6.00) group. When compared to the EPS group, there was a significant increase (*P < 0.05) in IL-4 in the EPS + VD + VA (62.00 ± 4.899) group. There was no significant difference between the EPS group and the EPS + D2 (54.00 ± 5.10) group.

Interleukin-13 (IL-13) levels in Motor cortex and Basal nuclei

Shows a significant decrease (**P < 0.01) in IL-13 in the EPS (39 ± 5.10) group when compared to the control group (108 ± 12.41). When compared to the EPS group, both EPS + VD + VA (94 + 10.77) and EPS + D2 (88.00 ± 10.68) groups show significant increases (**P < 0.01 and *P < 0.05, respectively) in IL-13 levels.

3.3 Concentration of Dopamine in M1 and CPu

When compared with the control group (9600 ± 509.9), EPS (5000 ± 447.2) shows a significant (***P < 0.001) decline in the concentration of dopamine. There was no significant difference between the control group and the EPS + VD + VA and EPS + VA groups. When compared with the EPS group, EPS + VD + VA (7400 ± 509.9) (*P < 0.05) and EPS + D2 (10000 ± 707.1) (***P < 0.001) show a significant increase in the level of dopamine.

3.4 Astrocytes Activation in Motor cortex

The EPS group (32.80 ± 2.083) showed a significant (***P < 0.001) increase in the expression of reactive astrocytes compared to the control group (12.20 ± 1.772). There was no significant increase in activated astrocytes between the control, EPS + VD + VA, and EPS + D2 groups. When compared with the EPS group, both EPS + VD + VA (15.80 ± 2.289) and EPS + D2 (19.20 ± 1.463) recorded significant (***P < 0.001) declines in the expression of reactive astrocytes.

3.5 Activation of Astrocytes in Striatum

Compared to the control group (7.80 ± 1.88), the EPS group (13.00 ± 0.95) exhibited a significant (*P < 0.05) increase in the expression of reactive astrocytes. However, both the EPS + VD + VA (8.20 ± 0.86) and EPS + D2 (10.20 ± 0.80) groups showed no significant increase in the expression of reactive astrocytes when compared to the control group.

Astrocytes are integral glial cells crucial for maintaining the stability of the central nervous system. They undergo rapid functional activation in response to various diseased or traumatic events [32]. In both physiological and pathological conditions, activated astrocytes play a key role in synthesizing and releasing neuroactive substances, including neurotrophic factors and inflammatory cytokines [33].

In this study, a significant increase in the expression of activated astrocytes was observed in the corticobasal loop, encompassing the motor cortex and striatum, of the EPS model. The corticobasal loop is primarily associated with the learning of motor skills and the execution of motor commands [34]. The activated astrocytes in this region exhibited characteristic features such as hypertrophy, an increase in size and proliferation, indicating an elevated number.

Activation of astrocytes in the corticobasal loop is a neuropathological hallmark of Parkinson's disease (PD) [35]. This suggests that our EPS model replicates the pathognomonic traits of PD, demonstrating good construct validity. Post-mortem examinations of Parkinson’s disease patients' brains revealed a negative correlation between neuronal death and the number of activated astrocytes [36, 37]. Therefore, the substantial increase in the expression of activated astrocytes indicates significant neurodegeneration in the corticobasal loop resulting from prolonged inhibition of the dopamine-2 receptor used in developing the EPS model.

Interestingly, the groups that received dopamine-2 agonists or vitamin D with vitamin A during the development of the EPS model did not show a significant increase in the expression of activated astrocytes. This suggests a potential neuroprotective effect of dopamine agonists and the combination of vitamin D with vitamin A. Previous studies have indicated that a deficiency in vitamin D may increase the risk of neurodegenerative pathologies [38, 39]. Additionally, dopamine agonists are known to provide neuroprotective effects by actively eliminating free radicals, enhancing the activities of radical-scavenging enzymes, and improving neurotrophic activity [40–43]. Both vitamin D and A are recognized for their antioxidant properties [44, 45], suggesting a potential shared neuroprotective mechanism. Furthermore, our previous findings have highlighted that the concurrent administration of vitamin D with vitamin A leads to a reduction in malondialdehyde (MDA) and superoxide dismutases, essential components in oxidative stress processes.

Astrocyte activation has been implicated in the generation of a significant amount of reactive oxygen species (ROS) through both the mitochondrial and NADPH oxidase pathways, leading to oxidative damage in various brain regions [46–48]. Consequently, the observed reduction in activated astrocyte density in the Vitamins treatment group could be linked to a decrease in oxidative stress and the associated damage in the cortico-basal loop.

Research has indicated a correlation between an increase in the expression of activated astrocytes and inflammatory cytokines [49–51]. Pro-inflammatory cytokines, predominantly produced by activated macrophages, play a pivotal role in regulating inflammatory responses. In the EPS model, there was a significant increase in the levels of pro-inflammatory cytokines, particularly TNF-α and IL-6, along with a significant decrease in the levels of anti-inflammatory cytokines IL-4 and IL-13. Thus, TNF-α, IL-6, IL-4, and IL-13 are implicated in the pathogenesis of EPS. Elevated levels of TNF-α and IL-10 are known to trigger the expression of reactive astrocytes, leading to cell death and tissue degeneration [52–55]. Recent studies have unraveled molecular mechanisms, including the activity of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines that modulate transcriptional programs in astrocytes associated with degenerative or protective functions [56–58].

The well-established evidence that Vitamin D3 therapy can reduce inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokine expression in an MPTP-induced Parkinson's disease model [59–62] supports the hypothesis that the observed decline in activated astrocyte density in the Vitamins treatment group may be linked to the anti-inflammatory and neuroprotective effects of vitamin D and A. This provides additional support for the potential therapeutic role of these vitamins in mitigating neurodegenerative processes associated with Extrapyramidal Syndromes (EPS).

In the current study, the EPS model group exhibited a significant decline in the concentration of dopamine, while the group treated with a vitamin D3 receptor agonist (VDRA) showed a significant improvement in dopamine concentration. Although the exact mechanism by which vitamin D3 receptor activation promotes the recovery of dopaminergic release is not fully understood, existing evidence suggests its potential involvement in affecting calcium metabolism, apoptosis, immunomodulation, and the upregulation of neurotrophins [63]. The findings from this study further suggest a role for the VDRA in regulating dopamine levels through the suppression of pro-inflammatory cytokines and the elevation of anti-inflammatory cytokines associated with astrocyte activation. Therefore, the reduced dopamine levels observed in the EPS model group could be linked to inflammatory processes, similar to what has been established in the substantia nigra of Parkinson's disease patients.

Extrapyramidal syndrome induces the expression of reactive astrocytes, a surge in pro-inflammatory cytokines, a decline in anti-inflammatory cytokines, as well as dopamine concentration in the corticobasal loop. The administration of vitamin D3 and A was found to exert its effect by suppressing pro-inflammatory cytokines and elevating anti-inflammatory cytokines associated with astrocyte activation. This suggests a potential therapeutic role for vitamin D3 and A in mitigating the neuroinflammatory processes and neurodegenerative changes associated with Extrapyramidal Syndromes.

Ethical Approval

All protocols for handling and caring for the animals adhered strictly to the guidelines set forth by the National Health Institute (NIH) and the Institutional Animal Care and Use Committee (IACUC). Ethical approval for the animal care and experimental procedures was obtained from the Animal Care and Use Research Ethics Committee (ACUREC) of Bayero University, Kano, Nigeria.

Funding

The authors conducted this research using only their own funding, without receiving any external grants or financial support from grant bodies for the study.

Availability of data and Materials

The datasets used and/or analysed during the current study is available with the corresponding author upon reasonable request.

Disclosure statement

No potential conflict of interest was reported by the author(s).

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Synergistic Action of Vitamin D3 and A Protects Motor Activity by Regulating Reactive Astrocytes, Inflammatory Cytokines, and Dopaminergic Activity in the Corticobasal Loop of a Mice Model of Extrapyramidal Syndrome

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Abstract

Figures

1.0 Introduction

2.0 Materials and Methods

2.1 Experimental animals

2.1.1 Experimental Design:

2.1.2 Housing and Nutrition:

2.1.3 Animal Welfare Protocols:

2.2 Animal treatment

2.3 Animal Sacrifice

2.4 Biochemical Analysis

2.5.1 Determination of Cytokines

2.5.2 Dopamine concentration

2.5.3 Immunohistochemistry

2.6 Photomicrography and Cell count:

2.7 Statistical Analysis

3.0 Results

3.1 ELISA analysis of pro-inflammatory cytokine levels

3.2 ELISA analysis of Anti-inflammatory cytokine levels

3.3 Concentration of Dopamine in M1 and CPu

3.4 Astrocytes Activation in Motor cortex

3.5 Activation of Astrocytes in Striatum

4.0 Discussion

Conclusion

Declarations

References

Additional Declarations

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