Hemodynamic changes and neuronal damage detected by 9.4 T MRI in rats with chronic cerebral ischemia and cognitive impairment.

INTRODUCTION: The bilateral common carotid artery occlusion (BCCAO) rat model is an ideal animal model for simulating the pathology of chronic brain hypoperfusion in humans. However, dynamic changes in neuronal activity, cellular edema, and neuronal structural integrity in vivo after BCCAO have rarely been reported. The purpose of this study is to use a 9.4 T MRI to explore the pathophysiological mechanisms of vascular dementia. MATERIALS AND METHODS: Twelve Sprague-Dawley (SD) rats were randomly divided into two groups: the sham group and the model group (n = 6 for each group). Rats were subjected to MRI using T2*WI, diffusion tensor imaging (DTI), and DWI sequences by MRI at the following six time points: presurgery and 6 h, 3 days, 7 days, 21 days, and 28 days postsurgery. Then, the T2*, fractional anisotropy (FA), and average apparent diffusion coefficient (ADC) values were measured in the bilateral cortices and hippocampi. After MRI scanning, all rats in both groups were subjected to the Y-maze test, novel object recognition test, and open-field test to assess their learning, memory, cognition, and locomotor activity. RESULTS: The T2*, FA, and ADC values in the cerebral cortex and hippocampus decreased sharply at 6 h after BCCAO in the model group compared with those of the sham group. By Day 28, the T2* and ADC values gradually increased to close to those in the sham group, but the FA values changed little, and the rats in the model group had worse learning, memory, and cognition and less locomotor activity than the rats in the sham group. CONCLUSIONS: The BCCAO is an ideal rat model for studying the pathophysiological mechanisms of vascular dementia.

Nephroprotective Effects of Tetramethylpyrazine Nitrone TBN in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal failure, but therapeutic options for nephroprotection are limited. Oxidative stress plays a key role in the pathogenesis of DKD. Our previous studies demonstrated that tetramethylpyrazine nitrone (TBN), a novel nitrone derivative of tetramethylpyrazine with potent free radical-scavenging activity, exerted multifunctional neuroprotection in neurological diseases. However, the effect of TBN on DKD and its underlying mechanisms of action are not yet clear. Herein, we performed streptozotocin-induced rat models of DKD and found that TBN administrated orally twice daily for 6 weeks significantly lowered urinary albumin, N-acetyl-ß-D-glycosaminidase, cystatin C, malonaldehyde, and 8-hydroxy-2'-deoxyguanosine levels. TBN also ameliorated renal histopathological changes. More importantly, in a nonhuman primate model of spontaneous stage III DKD, TBN increased the estimated glomerular filtration rate, decreased serum 3-nitrotyrosine, malonaldehyde and 8-hydroxy-2'-deoxyguanosine levels, and improved metabolic abnormalities. In HK-2 cells, TBN increased glycolytic and mitochondrial functions. The protective mechanism of TBN might involve the activation of AMPK/PGC-1α-mediated downstream signaling pathways, thereby improving mitochondrial function and reducing oxidative stress in the kidneys of DKD rodent models. These results support the clinical development of TBN for the treatment of DKD.

Therapeutic efficacy of novel memantine nitrate MN-08 in animal models of Alzheimer's disease.

Alzheimer's disease (AD) is a leading cause of dementia in elderly individuals and therapeutic options for AD are very limited. Over-activation of N-methyl-D-aspartate (NMDA) receptors, amyloid ß (Aß) aggregation, a decrease in cerebral blood flow (CBF), and downstream pathological events play important roles in the disease progression of AD. In the present study, MN-08, a novel memantine nitrate, was found to inhibit Aß accumulation, prevent neuronal and dendritic spine loss, and consequently attenuate cognitive deficits in 2-month-old APP/PS1 transgenic mice (for a 6-month preventative course) and in the 8-month-old triple-transgenic (3×Tg-AD) mice (for a 4-month therapeutic course). In vitro, MN-08 could bind to and antagonize NMDA receptors, inhibit the calcium influx, and reverse the dysregulations of ERK and PI3K/Akt/GSK3ß pathway, subsequently preventing glutamate-induced neuronal loss. In addition, MN-08 had favorable pharmacokinetics, blood-brain barrier penetration, and safety profiles in rats and beagle dogs. These findings suggest that the novel memantine nitrate MN-08 may be a useful therapeutic agent for AD.

Dual-Functional MN-08 Attenuated Pulmonary Arterial Hypertension Through Vasodilation and Inhibition of Pulmonary Arterial Remodeling.

[Figure: see text].

The Tetramethylpyrazine Analogue T-006 Alleviates Cognitive Deficits by Inhibition of Tau Expression and Phosphorylation in Transgenic Mice Modeling Alzheimer's Disease.

T-006, a small-molecule compound derived from tetramethylpyrazine (TMP), has potential for the treatment of neurological diseases. In order to investigate the effect of T-006 prophylactic treatment on an Alzheimer's disease (AD) model and identify the target of T-006, we intragastrically administered T-006 (3 mg/kg) to Alzheimer's disease (AD) transgenic mice (APP/PS1-2xTg and APP/PS1/Tau-3xTg) for 6 and 8 months, respectively. T-006 improved cognitive ability after long-term administration in two AD mouse models and targeted mitochondrial-related protein alpha-F1-ATP synthase (ATP5A). T-006 significantly reduced the expression of phosphorylated-tau, total tau, and APP while increasing the expression of synapse-associated proteins in 3xTg mice. In addition, T-006 modulated the JNK and mTOR-ULK1 pathways to reduce both p-tau and total tau levels. Our data suggested that T-006 mitigated cognitive decline primarily by reducing the p-tau and total tau levels in 3xTg mice, supporting further investigation into its development as a candidate drug for AD treatment.

Tetramethylpyrazine nitrone improves motor dysfunction and pathological manifestations by activating the PGC-1α/Nrf2/HO-1 pathway in ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons that results in skeletal muscle atrophy, weakness and paralysis. Oxidative stress plays a key role in the pathogenesis of ALS, including familial forms of the disease arising from mutation of the gene coding for superoxide dismutase (SOD1). We have used the SOD1G93A ALS mouse model to investigate the efficacy of 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a novel tetramethylpyrazine derivative armed with a powerful free-radical scavenging nitrone moiety. TBN was administered to mice by intraperitoneal or intragastric injection after the onset of motor deficits. TBN slowed the progression of motor neuron disease as evidenced by improved motor performance, reduced spinal motor neuron loss and the associated glial response, and decreased skeletal muscle fiber denervation and fibrosis. TBN treatment activated mitochondrial antioxidant activity through the PGC-1α/Nrf2/HO-1 pathway and decreased the expression of human SOD1. These findings suggest that TBN holds promise as a therapeutic agent for ALS.

Pharmacological Characterizations of anti-Dementia Memantine Nitrate via Neuroprotection and Vasodilation in Vitro and in Vivo.

We have previously designed and synthesized a series of novel memantine nitrates, and some of them have shown neuroprotective effects; however, the detailed mechanisms remain unknown. In this study, we demonstrated that MN-12, one of the memantine nitrates, concentration-dependently protected against glutamate-induced neurotoxicity in rat primary cultured cerebellar granule neurons (CGNs). Western blotting assays revealed that MN-12 might possess neuroprotective effects through the inhibition of ERK pathway and activation of PI3K/Akt pathway concurrently. Moreover, MN-12 concentration-dependently dilated precontracted rat middle cerebral artery through activation of NO-cGMP pathway ex vivo. In the 2-vessel occlusion (2VO) rat model, MN-12 alleviated the impairments of spatial memory and motor dysfunction possibly via neuroprotection and improvement of the cerebral blood flow. Furthermore, the results of preliminary pharmacokinetic studies showed that MN-12 might quickly distribute to the major organs including the brain, indicating that MN-12 could penetrate the blood-brain barrier. Taken together, MN-12 might provide multifunctional therapeutic benefits for dementia associated with Alzheimer's disease, vascular dementia, and ischemic stroke, via neuroprotection and vessel dilation to improve the cerebral blood flow.

The dual-functional memantine nitrate MN-08 alleviates cerebral vasospasm and brain injury in experimental subarachnoid haemorrhage models.

BACKGROUND AND PURPOSE: Cerebral vasospasm and neuronal apoptosis after subarachnoid haemorrhage (SAH) is the major cause of morbidity and mortality in SAH patients. So far, single-target agents have not prevented its occurrence. Memantine, a non-competitive NMDA re3ceptor antagonist, is known to alleviate brain injury and vasospasm in experimental models of SAH. Impairment of NO availability also contributes to vasospasm. Recently, we designed and synthesized a memantine nitrate MN-08, which has potent dual functions: neuroprotection and vasodilation. Here, we have tested the therapeutic effects of MN-08 in animal models of SAH. EXPERIMENTAL APPROACH: Binding to NMDA receptors (expressed in HEK293 cells), NO release and vasodilator effects of MN-08 were assessed in vitro. Therapeutic effects of MN-08 were investigated in vivo, using rat and rabbit SAH models. KEY RESULTS: MN-08 bound to the NMDA receptor, slowly releasing NO in vitro and in vivo. Consequently, MN-08 relaxed the pre-contracted middle cerebral artery ex vivo and increased blood flow velocity in small vessels of the mouse cerebral cortex. It did not, however, lower systemic blood pressure. In an endovascular perforation rat model of SAH, MN-08 improved the neurological scores and ameliorated cerebral vasospasm. Moreover, MN-08 also alleviated cerebral vasospasm in a cisterna magna single-injection model in rabbits. MN-08 attenuated neural cell apoptosis in both rat and rabbit models of SAH. Importantly, the therapeutic benefit of MN-08 was greater than that of memantine. CONCLUSION AND IMPLICATIONS: MN-08 has neuroprotective potential and can ameliorate vasospasm in experimental SAH models.

Tetramethylpyrazine Nitrone Reduces Oxidative Stress to Alleviate Cerebral Vasospasm in Experimental Subarachnoid Hemorrhage Models.

Cerebral vasospasm is one of the deleterious complications after subarachnoid hemorrhage (SAH), leading to delayed cerebral ischemia and permanent neurological deficits or even death. Free radicals and oxidative stress are considered as crucial causes contributing to cerebral vasospasm and brain damage after SAH. Tetramethylpyrazine nitrone (TBN), a derivative of the clinically used anti-stroke drug tetramethylpyrazine armed with a powerful free radical scavenging nitrone moiety, has been reported to prevent brain damage from ischemic stroke. The present study aimed to investigate the effects of TBN on vasospasm and brain damage after SAH. Two experimental SAH models were used, a rat model by endovascular perforation and a rabbit model by intracisternal injection of autologous blood. The effects of TBN on SAH were evaluated assessing basilar artery spasm, neuronal apoptosis, and neurological deficits. TBN treatment significantly attenuated vasospasm, improved neurological behavior functions and reduced the number of apoptotic neurons in both the SAH rats and rabbits. Mechanistically, TBN suppressed the increase in 3-nitrotyrosine and 8-hydroxy-2-deoxyguanosine immuno-positive cells in the cortex of SAH rat brain. Western blot analyses indicated that TBN effectively reversed the altered expression of Bcl-2, Bax and cytochrome C, and up-regulated nuclear factor erythroid-derived 2-like 2 (Nrf2) and hemeoxygenase-1 (HO-1) protein expressions. In the in vitro studies, TBN inhibited H2O2-induced bEnd.3 cell apoptosis and reduced ROS generation. Additionally, TBN alleviated the contraction of rat basilar artery rings induced by H2O2 ex vivo. In conclusion, TBN ameliorated SAH-induced cerebral vasospasm and neuronal damage. These effects of TBN may be attributed to its anti-oxidative stress effect and up-regulation of Nrf2/HO-1.

Novel neuroprotective tetramethylpyrazine analog T-006 promotes neurogenesis and neurological restoration in a rat model of stroke.

Neuronal death is among the deleterious pathological changes that occur after cerebral ischemia and can lead to transient or permanent neurological deficits. The tetramethylpyrazine analog T-006 has been shown to be a multifunctional neuroprotective agent; however, its neuroprotective effect and mechanism of action have not been studied in ischemic stroke model rats. This study investigated the neuroprotective effects of T-006 in rat stroke model using a battery of behavioral and molecular biological tests. Results indicated that T-006 treatment significantly improved neurological function and behavior. Double immunofluorescence staining showed that T-006 visibly improved the number of NeuN/BrdU, Nestin/BrdU, and DCX/BrdU cells and induced neuronal regeneration. Western blot analyses indicated that T-006 upregulated neurogenesis-related protein expression of postsynaptic density protein 95, brain-derived neurotrophic factor, synaptophysin, and myelin basic protein. Collectively, these data suggest that T-006 stimulated neurogenesis in rats with middle cerebral artery occlusion and restored neurological functions.

Miconazole stimulates post-ischemic neurogenesis and promotes functional restoration in rats.

Miconazole, a frequently used antifungal drug, has been identified with new functions to promote oligodendrocyte progenitor cells differentiation and to enhance remyelination. However, the neuroregenerative and therapeutic benefit of miconazole on ischemic stroke model have not been tested. In the present study, the effects of miconazole on a rat model of transient middle cerebral artery occlusion were evaluated. Rats received miconazole (10 mg/kg) or saline by intravenous administration for 7 days after stroke. A battery of neurobehavioral assessments, including rotarod test, open-field test, neurological severity score and novel object recognition task were evaluated. The results revealed a significant functional improvement in miconazole-treated rats compared with vehicle-treated control. Animals were sacrificed at 7 and 28 days after stroke. Double immunofluorescence staining for NeuN+/BrdU+, DCX+/BrdU+ and Nestin+/BrdU+ cells indicated miconazole significantly promoted neurogenesis. Western blotting analysis revealed miconazole upregulated the protein expression of brain derived neurotrophic factor, myocyte enhancer factor 2D, synaptophysin, and postsynaptic density protein 95, while downregulated the expression of cyclin-dependent kinase 5. Taken together, miconazole promoted functional recovery on ischemic stroke model via stimulating post-ischemic neurogenesis.

Neuroprotective Effect and Mechanism of Action of Tetramethylpyrazine Nitrone for Ischemic Stroke Therapy.

Our previous studies demonstrated that the multifunctional agent TBN, a derivative of tetramethylpyrazine armed with a nitrone moiety, displayed high therapeutic efficacy in experimental ischemic stroke models. However, its molecular mechanisms of action underlying the neuroprotective effect need further exploration. In the present study, we found that TBN had significant activities scavenging free radicals such as ·OH, O 2·- and ONOO-, inhibiting Ca2+ overload, maintaining mitochondrial function and preventing neuronal damage in primary cortical cultures. Further, TBN was effective in reducing brain infarction and ameliorating impairment of behavioral functions in the permanent middle cerebral artery occlusion (p-MCAo) rat model. TBN down-regulated the expression of pro-apoptotic factors Bax, while up-regulated the expression of anti-apoptotic factor Bcl-2 and increased the expression of pro-survival factors including p-Akt and p-GSK3ß in the peri-infarct cortex of p-MCAo rats. In addition, LY-294002 (a PI3K inhibitor) and MK2206 (an Akt inhibitor) significantly blocked the protective effect of TBN against OGD-induced death of cortical neurons. Taken together, the multifunctional mechanisms including scavenging free radicals, blocking calcium overload, maintaining mitochondrial function and activating the PI3K/Akt/p-GSK3ß cell survival pathway were possibly involved in the neuroprotective effects of TBN, making it a promising clinical candidate for the treatment of ischemic stroke.

Tetramethylpyrazine nitrone activates the BDNF/Akt/CREB pathway to promote post-ischaemic neuroregeneration and recovery of neurological functions in rats.

BACKGROUND AND PURPOSE: Neuronal regeneration from endogenous precursors is an attractive strategy for the treatment of ischaemic stroke. However, most stroke-generated newborn neurons die over time. Therefore, a drug that is both neuroprotective and pro-neurogenic may be beneficial after stroke. Here, we assessed the neurogenic and oligodendrogenic effects of tetramethylpyrazine nitrone (TBN), a neuroprotective drug candidate for stroke, in a rat model of ischaemic stroke. EXPERIMENTAL APPROACH: We used Sprague Dawley rats with middle cerebral artery occlusion (MCAO). TBN was administered by tail vein injection beginning at 3 h post ischaemia. Therapeutic effect of TBN was evaluated by neurological behaviour and cerebral infarction. Promotion of neurogenesis and oligodendrogenesis was determined by double immunofluorescent staining and Western blotting analyses. Primary cultures of cortical neurons were used to assess the effect of TBN on neuronal differentiation in vitro. KEY RESULTS: TBN reduced cerebral infarction, preserved and/or restored neurological function and promoted neurogenesis and oligodendrogenesis in rats after MCAO. In addition, TBN stimulated neuronal differentiation on primary culture of cortical neurons in vitro. Pro-neurogenic effects of TBN were attributed to its activation of the AKT/cAMP responsive element-binding protein through increasing brain-derived neurotrophic factor (BDNF) expression, as shown by the abolition of the effects of TBN by a specific inhibitor of BDNF receptor ANA-12 and by the PI3K inhibitor LY294002. CONCLUSION AND IMPLICATIONS: As TBN can simultaneously provide neuroprotection and pro-neurogenic effects, it may be a promising treatment for both acute phase neuroprotection and long-term functional recovery after ischaemic stroke.

Neuroprotective and axonal outgrowth-promoting effects of tetramethylpyrazine nitrone in chronic cerebral hypoperfusion rats and primary hippocampal neurons exposed to hypoxia.

Chronic cerebral hypoperfusion is an important risk factor for vascular dementia and other brain dysfunctions, for which there are currently no effective medications available. We investigated the neuroprotective and axonal outgrowth promoting effects of tetramethylpyrazine nitrone (TBN) in a permanent bilateral occlusion of the common carotid arteries (2VO) rat model and in primary hippocampal neurons exposed to oxygen glucose deprivation (OGD). At 6th week after 2VO, TBN increased the time spent in novel arms in the Y-maze test and improved the discrimination ratio in object reorganization task. TBN attenuated axonal damage, and reduced oxidative DNA injury and lipid peroxidation in white matter. TBN also attenuated the neuronal apoptosis and ameliorated accumulation of astrocytes in parietal cortex and CA1 region of hippocampus. Western blot analyses indicated that TBN increased Bcl-2 expression, decreased Bax and Caspase 3 expressions, and upregulated the phosphorylation levels of high-molecular weight neurofilament (p-NFH), Akt (p-Akt) and glycogen synthase kinase-3ß (p-GSK3ß) in hippocampus at 6th week after chronic hypoperfusion. In vitro, TBN rescued hippocampal neuronal viability and axonal elongation from OGD damage. The p-Akt and p-GSK3ß upregulation by TBN was abolished by a specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002, resulting in suppression of axonal outgrowth. Collectively, the results showed that TBN alleviated white matter lesion and impairment of cortex and hippocampus, attenuated oxidative damage and enhanced axonal outgrowth through the regulation of PI3K/Akt/GSK3ß signaling pathway, leading to improved cognitive deficit in a rat chronic hypoperfusion model.