In this study, we used the 2-VO model to investigate the stereoselective pharmacological activities of oxiracetam. We demonstrated for the first time that (S)-oxiracetam, but not (R)-oxiracetam, was the active ingredient in oxiracetam that alleviated the impairments in learning and memory, ameliorated the pathological damages, and increased the cerebral blood flow in the 2-VO rats. Its nootropic effects could be related to the inhibition of astrocyte activation in the hippocampus CA1 region during the chronic phase. During the acute phase in the 2-VO rats, (S)-oxiracetam acts by rescuing the abnormal metabolism of ATP and increasing the content of glutamate, glutamine and other small molecules in the cortex region.

Previous animal experiments have demonstrated that chronic cerebral hypoperfusion could induce impairments in learning and memory in 2-VO rats29. The Morris water maze is most frequently used to measure the spatial learning capacity in rats undergoing chronic cerebral hypoperfusion. In this study, using the Morris water maze test, we found that only (S)-oxiracetam and oxiracetam ameliorated the impairments in learning and memory (decreased escape latency and increased platform crossing) induced by the 2-VO surgery. This finding was consistent with a previous study in which (S)-oxiracetam was shown to be the active ingredient in oxiracetam that reversed the cognitive impairments induced by scopolamine. In this study, a dose-dependent effect of (S)-oxiracetam was not obvious, which may be due to the large individual variability in the behavioral test. In addition, the absorption of (S)-oxiracetam may be saturated at high doses30, which might have influenced the nootropic effects of (S)-oxiracetam.

Chronic cerebral hypoperfusion can result in severe neuronal damage in the hippocampus, cerebral cortex, and white matter (WM). Seven weeks after the 2-VO surgery, neuronal damage, including neuronal loss, shrinkage, and dark staining, was observed in the hippocampus and cortex. White matter lesions, including vacuolation and a disarrangement of the myelin fibers in the optical tract, were also detected. Our results showed that (S)-oxiracetam, but not (R)-oxiracetam, ameliorated the neuronal damage and white matter lesions. These histological changes were consistent with the cognitive impairments revealed by the Morris water maze test.

The cerebral blood flow plays an important role in maintaining normal brain functioning. A decreased cerebral blood flow was observed in dementia patients. Previous studies have shown that piracetam can increase the cerebral blood flow both in ischemic cats31, 32 and post-stroke aphasic patients33. In addition, piracetam can reduce the erythrocyte adhesion to the vascular endothelium, hinder vasospasm, and facilitate microcirculation34. Our results showed that the S-isomer increased the cerebral blood flow after the 2-VO surgery. However, (R)-oxiracetam had no such beneficial effects, indicating that (S)-oxiracetam, but not (R)-oxiracetam, was the active ingredient in oxiracetam that increased the cerebral blood flow in the chronic cerebral hypoperfused rats. Chronic reduction in cerebral blood flow in the adult rat is associated with a late-emerging CA1 cell loss and memory dysfunction. CA1 neurons begin to degenerate after several weeks of reduced energy availability due to the 2-VO, and this degeneration impairs memory. Since reduced neuronal energy metabolism is associated with the progressive neurodegeneration in disorders, such as Alzheimer’s disease29, the effect of oxiracetam on cerebrovascular impairment was investigated in rats. Oxiracetam administered after the re-perfusion at a dose of 100 mg/kg (i.v.) accelerated recovery. Oxiracetam is thought to have a protective effect against cerebrovascular impairment24. The improved cerebral blood flow after the (S)-oxiracetam treatment may be caused by the increased neuronal energy metabolism such as ATP metabolism and its protective effect in the cerebrovascular impairment. However, the specific mechanisms require further investigation.

Astrocyte activation is one of the major producers of inflammatory mediators in the nervous system35. Previous studies have shown that astrocytes are activated during the chronic phase36 of the 2-VO model. Activated astrocytes can cause neuronal damage and a blood brain barrier dysfunction by releasing neurotoxic molecules, including pro-inflammatory factors, reactive oxygen species, and reactive nitrogen species37, 38. In the 2-VO model, the astrocyte activation is closely associated with the cognitive impairments39, 40, and the control of astrocyte activation is likely an important therapeutic target41, 42. In addition, astrocytes play an important role in the dynamic regulation of the cerebral circulation43. In this study, we found that many reactive astrocytes that were labeled with GFAP were activated in the hippocampus CA1 region. In addition, (S)-oxiracetam alone inhibited the astrocyte activation. These results suggested that the nootropic effects of (S)-oxiracetam in the 2-VO rats might be related to the inhibition of astrocyte activation.

To further explore the underlying mechanisms of (S)-oxiracetam’s nootropic effects during the acute phase, we investigated the spatial distribution changes in small molecules in the brain tissues using MALDI-MSI. MALDI-MSI has been extensively used to analyze small molecules in biological samples by providing direct 2D visualization of the metabolic profiles. In this study, we systematically investigated the changes in the spatial distribution of ATP and other small molecules during the acute phase of the 2-VO rat brain. Our results showed that (S)-oxiracetam decreased the abnormal accumulation of glucose and citric acid and increased ATP metabolism. In addition, we demonstrated for the first time that (S)-oxiracetam increased the Glutamate-Glutamine Cycle, Malate-Aspartate Shuttle and the content of anti-oxidants and maintained the homeostasis of Na+ and K+ in the cortex region of the 2-VO rats.

Oxiracetam can cross the blood-brain barrier and distribute in the septum, hippocampus, cerebral cortex, and striatum44. (S)-oxiracetam has distribution profiles similar to racemic oxiracetam45. In addition, it has both higher absorption and slower elimination rates than racemic oxiracetam30, suggesting that (S)-oxiracetam might have a more favorable pharmacokinetic profile that can decrease toxicology risks and clinical dosage when developed as a new drug30, 45. The cortex region is more vulnerable to cerebral ischemia than other regions of the brain46. In addition, the regions of ischemic stroke mainly appear in the right hemisphere of the human brain47, which could be attributed to the more frequent use of the right hand, which leads to an increased blood supply in the left hemisphere48, 49, thereby leaving the right hemisphere more vulnerable to ischemic stroke. Furthermore, it could be caused by the asymmetrical insula and parietal control of the autonomic nervous system and alterations in the norepinephrine levels47. In this study, we also observed that the ischemic regions were mainly located in the cortex region in the right rat brain, indicating that laterality might also exist in rats50, 51. These results were also consistent with a previous 2-OV study reported by Sónia Sá Santos et al., where only one side of the brain is affected in the representative figure52. And (S)-oxiracetam ameliorated the abnormal metabolism of small molecules in the right cortex region.

Glucose is the key source of energy in the brain, and citric acid is the first mediator of the Tricarboxylic acid (TCA) cycle. Previous studies have shown that oxiracetam increases glucose utilization in cerebral ischemic rats53. In this study, the MSI results showed that the levels of glucose and citric acid increased after the 2-VO surgery. Consistently with a previous study, we found that (S)-oxiracetam decreased the abnormal accumulation of glucose and citric acid in the cortex region.

Glucose aerobic oxidation coupled with phosphorylation is the major pathway of ATP generation. ATP can be catabolized to ADP, AMP, and eventually uric acid following two main catabolic routes54. The restricted oxygen and glucose delivery caused by the 2-VO surgery interrupted mitochondrial oxidative phosphorylation and ATP synthesis. In this study, ATP, ADP and AMP were decreased in the model group. A previous study has shown that oxiracetam increases the ATP content in cultured astrocytes26 and improves the ratio of brain adenosine triphosphate/adenosine diphosphate (ATP/ADP) in the brain, and our study found that (S)-oxiracetam and oxiracetam increased the content of ATP and its downstream products ADP and AMP. These results suggested that (S)-oxiracetam and oxiracetam could rescue the interrupted mitochondrial oxidative phosphorylation and subsequent ATP metabolism.

In this study, the MSI results showed that the levels of glucose and citric acid increased, whereas the levels of ATP, ADP, and AMP decreased after the 2-VO surgery. The increase in glucose caused by the 2-VO surgery was likely due to the lower glucose utilization in the brain as suggested by a previous report53. The increased levels of citric acid and decreased levels of downstream products, including glutamate and aspartate, could have been caused by the arrest of the TCA cycle at the step from citrate to α-ketoglutarate. Furthermore, previous studies have shown that mitochondrial enzymes, such as aconitase and 2-oxoglutarate dehydrogenase, were inactivated55, 56, and the activity of citrate synthase was unchanged after ischemia57.

Glutamate-mediated excitotoxicity plays an important role in the pathogenesis of cerebral ischemia58. L-Aspartate is another excitatory neurotransmitter that plays a key role in the malate-aspartate shuttle that transfers NADH in the cytosol through the inner membrane to the mitochondria for oxidative phosphorylation and ATP synthesis. N-acetylaspartate (NAA), which is an indicator of neuronal damage and mitochondrial dysfunction, is synthesized in the mitochondria from aspartate and acetyl-CoA by the enzyme L-aspartate N-acetyltransferase57. A previous study has shown that piracetam decreases the release of glutamate and aspartate in cortical cells upon oxygen and glucose deprivation. However, it was unknown whether oxiracetam had effects on glutamate and aspartate. In this study, the levels of glutamine, glutamate, aspartate, and N-acetylaspartate (NAA) decreased after the 2-VO surgery. The treatment with (S)-oxiracetam and oxiracetam increased the content of these metabolites, indicating that oxiracetam and (S)-oxiracetam also have effects on glutamate and aspartate.

During the ischemic period, the restricted delivery of oxygen impairs ATP synthesis and causes the continuous production of reactive oxygen species59, which can result in necrosis, apoptosis, and eventually neuronal death. Glutathione (GSH) and ascorbic acid (AA) are important anti-oxidants that can directly scavenge ROS in the brain. Although taurine is not a characteristic scavenger of ROS, it exhibits anti-oxidant activity through several indirect mechanisms, including reducing ROS production, limiting ROS activation, and interfering with ROS-producing inflammatory reactions60. Previous studies have reported that piracetam reverses the cognitive dysfunction and reduces the oxidative stress induced by propoxur and phosphamidon in the brain61, 62. However, it was unknown whether oxiracetam had anti-oxidative stress effects. In this study, the content of glutathione, ascorbic acid, and taurine decreased after the 2-VO surgery. The treatment with (S)-oxiracetam and oxiracetam increased the content of glutathione, ascorbic acid, and taurine in the cortex region, which is consistent with their anti-oxidant effects in which the SOD activity increased, and the MDA content decreased during the chronic phase (Supplementary Fig. 4).

Na+ and K+ are important inorganic ions in the brain. These ions are responsible for maintaining the membrane potential and are essential for neuronal activities. Na+- K+-ATPase and Na+-K+-2Cl−cotransporter (NKCC) are two important transporters/channels that maintain the intracellular and extracellular Na+ and K+ concentrations. After the occlusion of the cerebral artery, the supply of oxygen and glucose is not sufficient to power the Na/K ATPase. The increased concentration of Na+ and decreased concentration of K+ in the brain tissue result in necrosis63, 64. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is a glutamate ionotropic transmembrane receptor that mediates rapid synaptic transmission in the brain65. The principal ions fated by the AMPA receptor are Na+ and K+. Oxiracetam is reported to act as a positive allosteric modulator of the AMPA receptor66. However, whether oxiracetam has effects on Na+ and K+ is unknown. Our results showed that the concentration of Na+ increased, and the concentration of K+ decreased in the cortex region after the 2-VO surgery. The treatment with oxiracetam and (S)-oxiracetam rescued the abnormal ion content, which might have been caused by the increased activity of abluminal Na/K-ATPase, the decreased activity of luminal endothelial NKCC67, and the modulation of the AMPA receptor. However, the specific mechanisms require further investigation.

This is the first report describing the precise distribution of small molecules after (S)-oxiracetam treatment by MALDI-MSI during the acute phase of 2-VO. The MALDI-MSI technique can be successfully applied to the pharmacological evaluation and study of mechanisms. Although previous studies have investigate different aspects of the mechanisms of (S)-oxiracetam, these studies lack spatial distributing information of the analyzed substances. Using MALDI-MSI, we simultaneously observed 15 changed small molecules in the right cortex region of the rat brain. This finding allowed us obtain spatial distributing information regarding the changed small molecules after the drug treatment and a better understanding of the mechanisms of (S)-oxiracetam.

Of the 15 metabolites identified by MALDI-MSI, the following eight metabolites were also validated by LC-MS/MS: glutamine, glutamate, N-acetylaspartate, ascorbic acid, glutathione, ADP, AMP, and GMP. However, the existence of glucose isomers and the degradation of ATP could result in the failure of their validation using LC-MS/MS. The eight metabolites identified by the two distinct methods were related to the ATP metabolism pathway, the glutamate-glutamine cycle and anti-oxidants, which is indicative of their contribution to the nootropic effects of (S)-oxiracetam. ATP is the most direct source of energy in the human body. After an ischemic stroke, the decreased blood flow causes an ATP reduction and energy depletion and initiates excitotoxic mechanisms that are harmful to neurons. Glutamate is the major excitatory amino acid in the brain and plays an important role in the pathogenesis of cerebral ischemia. Ascorbic acid and glutathione are important anti-oxidants that can directly scavenge ROS in the brain. (S)-Oxiracetam increased ATP metabolism, the glutamate-glutamine cycle, the malate-aspartate shuttle, and the content of anti-oxidants. This finding indicates that (S)-oxiracetam could exert its nootropic effects by increasing the energy supply, inhibiting the toxicity of excitatory amino acids, increasing the content of anti-oxidants, and eventually reducing neuronal death.

Neuronal cell death occurs throughout the chronic cerebral hypoperfusion; during the acute phase of the 2-VO surgery, the blood flow disruptions rapidly limit the delivery of oxygen and glucose to neurons, thereby causing ATP reduction and energy depletion and initiating excitotoxic mechanisms that are deleterious to neurons65, 68. In turn, this process aggravates the reduction in glucose and ATP. Therefore, the decreased concentration of small molecules, such as ATP and glucose, observed after the 2VO-surgery could be due to the decreased blood flow and/or the increased neuronal death. Similarly, the increased concentration of small molecules after the (S)-oxiracetam treatment could be due to the increased cerebral blood flow and/or neuron survival. Oxiracetam acts as a positive modulator of AMPA-sensitive glutamate receptors in neurons66. This action increases the density of receptor binding sites for AMPA and calcium uptake66, 69. The AMPA receptor, which is another type of ionotropic channel, is known to mediate the rapid and immediate postsynaptic response to glutamate release and, thus, may contribute to synaptic plasticity70. So, the possibility that oxiracetam increases the glutamate concentration in the cortex region of 2-VO rats may be related to its positive regulation on the AMPA receptor. However, no studies have investigated whether (S)-oxiracetam increases the concentrations of small molecules by increasing neuronal survival and positive regulate AMPA receptor in the cortex region using the 2-VO model. Its detailed mechanism needs to be further explored in the future.

In conclusion, our study demonstrated that (S)-oxiracetam, but not (R)-oxiracetam, was the active ingredient in oxiracetam that alleviated the cognitive impairments in chronic cerebral hypoperfused rats. (S)-Oxiracetam exerted its nootropic effects by decreasing astrocyte activation and rescuing the abnormal metabolism of small molecules in the brain tissues during the acute phase in 2-VO rats. Therefore, (S)-oxiracetam alone might be a nootropic drug suitable for the treatment of dementia caused by chronic cerebral hypoperfusion.