Alpha-Lipoic Acid Alleviates Cerebral Ischemic Injury-Induced Cognitive Impairment and Alzheimer’s Disease-Related Pathologies Through Modulation of GSK-3β in a Rat Model of Transient Middle Cerebral Artery Occlusion

There are two main types of dementia: Alzheimer’s disease (AD) and vascular cognitive impairment and dementia (VCID). Alpha-lipoic acid (ALA) has antioxidant and anti-inammatory properties, and protects against cerebral ischemia-reperfusion (I/R) injury. We aimed to investigate the protective role of ALA in cerebral I/R injury-related cognitive impairment in a rat model of transient middle cerebral artery occlusion (tMCAO), which mimics VCID in humans.


Background
Dementia is a clinically challenging disease worldwide as we face an aging society [1]. Vascular cognitive impairment and dementia (VCID) is a major subtype of dementia resulting from a spectrum of cerebral vascular diseases [2], while Alzheimer's disease (AD) is a major subtype of dementia due to neurodegeneration [3,4]. Although the importance of VCID has received growing attention, the mechanisms related to the neuropathological changes that result in cognitive decline are complex, and effective treatment is still lacking [2]. It has been shown that cerebral ischemia could lead to AD-related pathologies [5].
Glycogen synthase kinase (GSK), a constitutive protein kinase, is not only involved in diverse physiological functions, but also in the pathogenesis of many diseases [6]. GSK-3β activation has been found in cerebral ischemic injury [7][8][9]. Inhibition of GSK-3β activation has been shown to protect against ischemic neuronal injury. In addition, GSK-3β has been shown to play a detrimental role in the pathogenesis of AD [10]. Its activation has been linked to Aβ production and hyperphosphorylation of tau.
Alpha-lipoic acid (ALA) is a compound that through multiple mechanisms protects against cerebral ischemic injury, including anti-oxidant and anti-in ammatory activities [11]. However, whether it is bene cial in reducing cerebral I/R injury-related cognitive impairment and AD-related pathologies is not clear. The purpose of this study was to test the hypothesis that ALA protects against cognitive impairment and AD-related pathologies after cerebral I/R injury in a rat model of transient middle cerebral artery occlusion (tMCAO) through modulation of GSK-3β.

Animals and Treatments
Adult, male Sprague-Dawley rats (280 to 320 g, BioLASCO Taiwan Co., Ltd., Taipei, Taiwan) were used in this study. The rats were housed in groups of 4 at an ambient temperature of 22 ± 1°C with a 12-h lightdark cycle according to the guidelines of the Institutional Animal Care and Use Committee at Chi Mei Medical Center (approval no. 104122916). Pellet rat chow and tap water were available ad libitum. The rats were anesthetized with Rompun and Zoletil 50 (Virbac of Brazil, Brazil) (0.01 and 0.04 mg/g of body weight, respectively). ALA (purity of >99%) was purchased from Sigma Biotechnology (St. Louis, MO, USA) and dissolved in 10% ethanol. Rats were randomly assigned to three experimental groups: sham, MCAO + vehicle (1 mg/kg), and MCAO + ALA (25 mg/kg). ALA and vehicle (10% ethanol) were administered intraperitoneally 120 minutes after the onset of cerebral ischemia.
tMCAO Model of Rats tMCAO was induced using the intraluminal lament procedure of Koizumi et al. and Longa et al. [12,13]. A midline cervical incision was made, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed. A length of 4-0 surgical nylon suture (20 to 21 mm) with a silicon rubber-coated head of 0.4 mm diameter (403956PK10; Doccol) [14] was inserted from the ECA into the lumen of the ICA to block the ow of middle cerebral artery (MCA). One hundred and twenty minutes after the completion of MCA occlusion, reperfusion was allowed by withdrawal of the suture thread until the tip cleared the ICA. Sham-operated animals underwent the same surgical procedure, in which suture was inserted without silicon rubber-coated head into the ICA.
Behavioral Evaluation of Neurological De cits -Modi ed Neurologic Severity Score (mNSS) Modi ed neurologic severity scores (mNSS) were used by an investigator who was blinded to the experimental groups to assess the severity of neurological de cits in rats after tMCAO. The mNSS was evaluated as previously described [15]. The mNSS consists of motor, sensory, balance, and re ex tests, and is graded from 0 to 18 (normal score 0; maximal de cit score 18) to evaluate the severity of injury.

Y Maze Test
The Y-maze test [16,17] was used to evaluate cognitive impairment in rats after tMCAO. The Y-shaped maze is composed of three white and opaque plastic arms (35 cm long, 25 cm high, and 10 cm wide) equally spaced at a 120° angle from each other. The rats were allowed to freely explore the three arms for 8 minutes. The rat movements inside the arms were recorded by a digital CCD camera mounted above the apparatus and were analyzed using the Top Scan system software (ver. 3.00; Clever Sys., Inc., VA, USA). The spontaneous alternation percentage (SA%) was calculated as the actual alternations/maximum alternations × 100.

The Measurements of Infarct Volumes and Brain Edema
Seventy-two hours after tMCAO, rats (n =8 for each group) were anesthetized and decapitated. Their brains were rapidly removed and sliced into six 2-mm-thick coronal sections. These slices were incubated in a solution of 2% 2, 3, 5-triphenyltetrazolium chloride (TTC, Sigma) in a water bath in the dark at 37°C for 30 minutes, and were then photographed separately using a digital camera (Canon, Japan). Infarct was de ned as unstained areas. The total infarct volume was measured in each slice and summed using computerized planimetry, as previously described [18,19]. For brain edema measurement, the percentage was calculated using the following formula : % brain edema = (the volume of ischemic hemisphere-the volume of the contralateral hemisphere)/the volume of the contralateral hemisphere) × 100.

Immuno uorescence Assay
Seven days after cerebral ischemia-reperfusion injury, rats were perfused transcardially with 0.9% NaCl. The brains were removed from the skulls and post-xed in 10% formaldehyde. Brain tissues were sectioned at a thickness of 4 μm according to the standard procedure. The sections were depara nized and hydrated gradually, as examined by immuno uorescence. Brie y, tissue sections were soaked in 0.01 M citrate buffer (pH 6.0), heated to the boiling point, blocked with 10% normal goat serum for 30 minutes at 37°C, and then incubated overnight at 4°C with mouse monoclonal antibody to activate microglia marker Ionized calcium-binding adaptor molecule 1 (Iba1) (1:200, Abcam), rabbit monoclonal antibody to

Statistical Analysis
All data are expressed as the mean ± standard error of the mean (SEM). Statistical comparisons were performed using the Kruskal-Wallis test, followed by post hoc comparisons. The Dunn method was used to compare more than two groups, while the Mann-Whitney U test was used for comparing two groups. P values less than 0.05 were considered as statistically signi cant differences.

Results
ALA alleviated severity of cerebral I/R injury and related neurologic de cits and cognitive impairment To investigate whether ALA can alleviate the severity of cerebral I/R injury, we measured the area of TTC stain and brain edema at 3 days, and neurobehavioral de cits 3 and 7 days after tMCAO. We found that ALA reduced the infarct area and brain edema (Fig. 1A, 1B, and 1C). ALA improved neurological de cits as measured by mNSS 3 and 7 days after MCAO (Fig. 1D).
To investigate whether ALA can alleviate cerebral I/R injury-related cognitive impairment, we measured the spontaneous alternation percentage in Y maze test 3, 7, and 28 days after tMCAO. As shown in Fig. 1E, rats receiving ALA showed a higher spontaneous alternation percentage in the Y-maze test than those receiving vehicle.
ALA alleviated activation of GSK-3β and microglia after cerebral I/R injury To evaluate GSK-3β activation after cerebral I/R injury, we measured the levels of phosphorylated GSK-3β by immuno uorescence staining in the rat hippocampus 7 days after tMCAO. ALA was found to signi cantly alleviate the activation of GSK-3β after cerebral I/R injury ( Fig. 2A, 2B). To assess the activation of microglia after cerebral I/R injury, we examined the levels of activated microglia marker Iba1 with immuno uorescence staining in the rat hippocampus 7 days after tMCAO. Furthermore, ALA signi cantly alleviated the activation of microglia after cerebral I/R injury (Fig. 2C, 2D).
ALA alleviated AD-related pathologies and maintained neuronal integrity after cerebral I/R injury To characterize the pattern of AD-related pathologies after cerebral I/R injury, we measured the expression of APP, BACE1, Aβ, and tau in the rat hippocampus using immuno uorescence staining 7 days after tMCAO. As shown in gure 3, the expression of the above-mentioned enzyme and proteins increased signi cantly in the rat hippocampus after cerebral I/R injury, which was signi cantly alleviated by the administration of ALA.
To evaluate the neuronal integrity after cerebral I/R injury, we measured the expression of synaptophysin in the rat hippocampus using immuno uorescence staining 7 days after tMCAO. As shown in gure 4, ALA maintained neuronal integrity by increasing the number of neuronal synapses and improving their alignment in the rat hippocampus after cerebral I/R injury.

Discussion
In this study, we demonstrated that ALA alleviated cognitive impairment and AD-related pathologies after cerebral ischemia-reperfusion injury in a rat model of tMCAO through down-regulation of GSK-3β. After transient cerebral ischemia for 2 h, cerebral infarction and brain edema were found, and consequent neurologic de cits and cognitive impairment were observed in tMCAO rats. Meanwhile, GSK-3β and microglia were activated in the hippocampus. Furthermore, increased expression of AD-related proteins (APP, BACE1, Aβ, and tau) and reduced expression of synaptophysin were found in the hippocampus 7 days after cerebral I/R injury. It was found that administration of ALA after cerebral I/R injury reduced cerebral infarction, brain edema, and improved neurologic de cits and cognitive impairment. In parallel with these ndings, ALA reduced the expression of APP, BACE1, Aβ, and phosphorylated tau in the hippocampus, down-regulated the activation of GSK-3β and microglia, and improved the integrity of neuronal synapses.
To the best of our knowledge, this is the rst report indicating that ALA can down-regulate the activation of GSK-3β after cerebral I/R injury and reduce AD-related pathologies, as we demonstrated in this study. It has been shown that cerebral hypoxia-ischemia can increase the production of APP and activate BACE1, and further increase the production of Aβ [5] and the levels of Tau proteins [5,20], suggesting that cerebral ischemia can produce AD-related pathologies, including activation of BACE1 and increased production of APP, Aβ, and Tau. Indeed, our ndings support this notion (Fig. 3).
ALA is an organosulfur compound displaying mainly anti-oxidant and anti-in ammatory activity [21,22]. The application of ALA in diverse diseases, including CNS diseases, has been receiving growing attention, [23][24][25]. Among them, ALA has long been recognized to be protective against cerebral I/R injury [25]. The mechanisms through which ALA protects against cerebral I/R injury involve mainly its anti-oxidant and anti-in ammatory activities. In the current study, we demonstrated the anti-in ammatory protective effects of ALA against the activation of GSK-3β and microglia that may lead to cognitive impairment and AD-related pathologies after cerebral I/R injury. In fact, activation of GSK-3β and microglia play an important role in the pathological consequences of cerebral I/R injury.
GSK is a constitutive protein kinase, with multiple function involving diverse pathophysiological pathways. Physiologically, it has been found to be involved in the regulation of cell cycle, gene expression, development, and metabolism [26]. Pathologically, it is involved in psychiatric diseases, neurological diseases, cardiovascular diseases, diabetes mellitus, in ammatory diseases, bone diseases, and cancer [6]. GSK-3β activation has been found in cerebral ischemic injury [7][8][9]. Application of GSK-3β inhibitors protects against ischemic neuronal injury. GSK-3β activation has been noted in the pathophysiological processes leading to AD [10,27]. Moreover, GSK-3β activation seems to be involved in the production of AD-related pathologies after cerebral ischemic injury. GSK-3β activation has been correlated with increased production of APP [28,29] and hyperphosphorylation of Tau [28] in the neonatal rat brain after hypoxia-ischemia. Furthermore, inhibition of GSK-3β decreased the production of APP [28] and hyperphosphorylation of Tau [28,30], and exerted its neuroprotective activity against cerebral I/R injury. In the current study, we clearly demonstrated that reduction in the activation of GSK-3β by ALA reduced AD-related pathologies, including activation of BACE1 and production of APP, Aβ, and Tau in cerebral I/R injury (Fig. 3).
Although not directly connected to cerebral ischemia, GSK-3β activation has been shown to lead to microglial activation in another in ammatory condition. Green and Nolan [31] have shown that lipopolysaccharide (LPS) can induce an increase in GSK-3β expression and activity in microglia in vitro. The current study showed that GSK-3β activation correlated well with microglia activation, since ALA down-regulated GSK-3β activation in parallel with microglia activation in cerebral I/R injury.
Microglial activation has been noted for a long time in cerebral ischemic injury [32]. The early activation of microglia in cerebral ischemic injury is detrimental to the brain. In our previous study, it was found that early administration of ALA can alleviate cerebral I/R injury in rats by down-regulating microglia activation [11]. In the current study, early administration of ALA not only alleviated cerebral I/R injury but also improved cognitive impairment and reduced AD-related pathologies through down-regulation of microglia activation.
Nevertheless, this is the rst study indicating that ALA can down-regulate the expression of GSK-3β after cerebral I/R injury. Furthermore, this is also the rst study indicating that ALA can reduce AD-related pathologies after cerebral I/R injury. In the current study, ALA was shown to down-regulate the activation of GSK-3β and microglia, and reduce AD-related pathologies, including the expression of APP, BACE1, Aβ, and tau after cerebral I/R injury.

Limitations
Our study is a descriptive study. It demonstrated the cascade of Alzheimer's disease-related neuropathological changes after cerebral ischemic injury in the rat model of transient middle cerebral artery occlusion. Future work is needed in mechanistic exploration regarding how ALA regulates GSK-3β after cerebral ischemic injury. Besides, this is a in vivo study and it warrants further efforts in future application of ALA in clinical treatment. In fact, ALA has been used as either the nutritional supplement or the drug in the treatment of diabetic polyneuropathy in many countries. Hence, the translational use of ALA in the treatment of VCID can be expected at least in terms of safety issues.

Conclusion
In summary, in addition to its role in AD pathology, GSK-3β might also be involved in VCID, which also shows AD-related pathologies after cerebral I/R injury. Importantly, AD and VCID account for the majority of dementia cases worldwide. In this study, we demonstrated that ALA can not only reduce the severity of cerebral infarction, brain edema, and neurobehavioral de cits, but also of AD-related pathologies, preserved the integrity of neuronal synapses, and improved cognitive impairment after cerebral I/R injury. Our study addresses the potential of ALA in the treatment of VCID after acute ischemic stroke in future clinical practice.