Tajinder S Dhammu

S-Nitrosoglutathione reduces oxidative injury and promotes mechanisms of neurorepair following traumatic brain injury in rats

BackgroundTraumatic brain injury (TBI) induces primary and secondary damage in both the endothelium and the brain parenchyma, collectively termed the neurovascular unit. While neurons die quickly by necrosis, a vicious cycle of secondary injury in endothelial cells exacerbates the initial injury in the neurovascular unit following TBI. In activated endothelial cells, excessive superoxide reacts with nitric oxide (NO) to form peroxynitrite. Peroxynitrite has been implicated in blood brain barrier (BBB) leakage, altered metabolic function, and neurobehavioral impairment. S-nitrosoglutathione (GSNO), a nitrosylation-based signaling molecule, was reported not only to reduce brain levels of peroxynitrite and oxidative metabolites but also to improve neurological function in TBI, stroke, and spinal cord injury. Therefore, we investigated whether GSNO promotes the neurorepair process by reducing the levels of peroxynitrite and the degree of oxidative injury.MethodsTBI was induced by controlled cortical impact (CCI) in adult male rats. GSNO or 3-Morpholino-sydnonimine (SIN-1) (50 μg/kg body weight) was administered orally two hours following CCI. The same dose was repeated daily until endpoints. GSNO-treated (GSNO group) or SIN-1-treated (SIN-1 group) injured animals were compared with vehicle-treated injured animals (TBI group) and vehicle-treated sham-operated animals (Sham group) in terms of peroxynitrite, NO, glutathione (GSH), lipid peroxidation, blood brain barrier (BBB) leakage, edema, inflammation, tissue structure, axon/myelin integrity, and neurotrophic factors.ResultsSIN-1 treatment of TBI increased whereas GSNO treatment decreased peroxynitrite, lipid peroxides/aldehydes, BBB leakage, inflammation and edema in a short-term treatment (4-48 hours). GSNO also reduced brain infarctions and enhanced the levels of NO and GSH. In a long-term treatment (14 days), GSNO protected axonal integrity, maintained myelin levels, promoted synaptic plasticity, and enhanced the expression of neurotrophic factors.ConclusionOur findings indicate the participation of peroxynitrite in the pathobiology of TBI. GSNO treatment of TBI not only reduces peroxynitrite but also protects the integrity of the neurovascular unit, indicating that GSNO blunts the deleterious effects of peroxynitrite. A long-term treatment of TBI with the same low dose of GSNO promotes synaptic plasticity and enhances the expression of neurotrophic factors. These results support that GSNO reduces the levels of oxidative metabolites, protects the neurovascular unit, and promotes neurorepair mechanisms in TBI.

The inhibitory effect of S-nitrosoglutathione on blood–brain barrier disruption and peroxynitrite formation in a rat model of experimental stroke

The hallmark of stroke injury is endothelial dysfunction leading to blood–brain barrier (BBB) leakage and edema. Among the causative factors of BBB disruption are accelerating peroxynitrite formation and the resultant decreased bioavailability of nitric oxide (NO). S‐nitrosoglutathione (GSNO), an S‐nitrosylating agent, was found not only to reduce the levels of peroxynitrite but also to protect the integrity of BBB in a rat model of cerebral ischemia and reperfusion (IR). A treatment with GSNO (3 μmol/kg) after IR reduced 3‐nitrotyrosine levels in and around vessels and maintained NO levels in brain. This mechanism protected endothelial function by reducing BBB leakage, increasing the expression of Zonula occludens‐1 (ZO‐1), decreasing edema, and reducing the expression of matrix metalloproteinase‐9 and E‐selectin in the neurovascular unit. An administration of the peroxynitrite‐forming agent 3‐morpholino sydnonimine (3 μmol/kg) at reperfusion increased BBB leakage and decreased the expression of ZO‐1, supporting the involvement of peroxynitrite in BBB disruption and edema. Mechanistically, the endothelium‐protecting action of GSNO was invoked by reducing the activity of nuclear factor kappa B and increasing the expression of S‐nitrosylated proteins. Taken together, the results support the ability of GSNO to improve endothelial function by reducing nitroxidative stress in stroke.

Stimulation of functional recovery via the mechanisms of neurorepair by S-nitrosoglutathione and motor exercise in a rat model of transient cerebral ischemia and reperfusion

PURPOSE Stroke disability stems from insufficient neurorepair mechanisms. Improvement of functions has been achieved through rehabilitation or therapeutic agents. Therefore, we combined exercise with a neurovascular protective agent, S-nitrosoglutathione (GSNO), to accelerate functional recovery. METHODS Stroke was induced by middle cerebral artery occlusion for 60 min followed by reperfusion in adult male rats. Animals were treated with vehicle (IR group), GSNO (0.25 mg/kg, GSNO group), rotarod exercise (EX group) and GSNO plus exercise (GSNO+EX group). The groups were studied for 14 days to determine neurorepair mechanisms and functional recovery. RESULTS Treated groups showed reduced infarction, decreased neuronal cell death, enhanced neurotrophic factors, and improved neurobehavioral functions. However, the GSNO+EX showed greater functional recovery (p < 0.05) than the GSNO or the EX group. A GSNO sub group, treated 24 hours after IR, still showed motor function recovery (p < 0.001). The protective effect of GSNO or exercise was blocked by the inhibition of Akt activity. CONCLUSIONS GSNO and exercise aid functional recovery by stimulating neurorepair mechanisms. The improvements by GSNO and exercise depend mechanistically on the Akt pathway. A combination of exercise and GSNO shows greater functional recovery. Improved recovery with GSNO, even administered 24 hours post-IR, demonstrates its clinical relevance.

Promoting endothelial function by S-nitrosoglutathione through the HIF-1α/VEGF pathway stimulates neurorepair and functional recovery following experimental stroke in rats

Background For stroke patients, stimulating neurorepair mechanisms is necessary to reduce morbidity and disability. Our previous studies on brain and spinal cord trauma show that exogenous treatment with the S-nitrosylating agent S-nitrosoglutathione (GSNO) – a nitric oxide and glutathione metabolite of the human body – stimulates neurorepair and aids functional recovery. Using a rat model of cerebral ischemia and reperfusion (IR) in this study, we tested the hypothesis that GSNO invokes the neurorepair process and improves neurobehavioral functions through the angiogenic HIF-1α/VEGF pathway. Methods Stroke was induced by middle cerebral artery occlusion for 60 minutes followed by reperfusion in adult male rats. The injured animals were treated with saline (IR group, n=7), GSNO (0.25 mg/kg, GSNO group, n=7), and GSNO plus the HIF-1α inhibitor 2-methoxyestra-diol (2-ME) (0.25 mg/kg GSNO + 5.0 mg/kg 2-ME, GSNO + 2-ME group, n=7). The groups were studied for either 7 or 14 days to determine neurorepair mediators and functional recovery. Brain capillary endothelial cells were used to show that GSNO promotes angiogenesis and that GSNO-mediated induction of VEGF and the stimulation of angiogenesis are dependent on HIF-1α activity. Results IR injury increased the expression of neurorepair mediators HIF-1α, VEGF, and PECAM-1 and vessel markers to a limited degree that correlate well with significantly compromised neurobehavioral functions compared with sham animals. GSNO treatment of IR not only remarkably enhanced further the expression of HIF-1α, VEGF, and PECAM-1 but also improved functioning compared with IR. The GSNO group also had a higher degree of vessel density than the IR group. Increased expression of VEGF and the degree of tube formation (angiogenesis) by GSNO were reduced after the inhibition of HIF-1α by 2-ME in an endothelial cell culture model. 2-ME treatment of the GSNO group also blocked not only GSNO’s effect of reduced infarct volume, decreased neuronal loss, and enhanced expression of PECAM-1 (P<0.001), but also its improvement of motor and neurological functions (P<0.001). Conclusion GSNO stimulates the process of neurorepair, promotes angiogenesis, and aids functional recovery through the HIF-1α-dependent pathway, showing therapeutic and translational promise for stroke.

Blocking a vicious cycle nNOS/peroxynitrite/AMPK by S-nitrosoglutathione: implication for stroke therapy

BackgroundStroke immediately sets into motion sustained excitotoxicity and calcium dysregulation, causing aberrant activity in neuronal nitric oxide synthase (nNOS) and an imbalance in the levels of nitric oxide (NO). Drugs targeting nNOS-originated toxicity may therefore reduce stroke-induced damage. Recently, we observed that a redox-modulating agent of the NO metabolome, S-nitrosoglutathione (GSNO), confers neurovascular protection by reducing the levels of peroxynitrite, a product of aberrant NOS activity. We therefore investigated whether GSNO-mediated neuroprotection and improved neurological functions depend on blocking nNOS/peroxynitrite-associated injurious mechanisms using a rat model of cerebral ischemia reperfusion (IR).ResultsIR increased the activity of nNOS, the levels of neuronal peroxynitrite and phosphorylation at Ser1412 of nNOS. GSNO treatment of IR animals decreased IR-activated nNOS activity and neuronal peroxynitrite levels by reducing nNOS phosphorylation at Ser1412. The Ser1412 phosphorylation is associated with increased nNOS activity. Supporting the notion that nNOS activity and peroxynitrite are deleterious following IR, inhibition of nNOS by its inhibitor 7-nitroindazole or reducing peroxynitrite by its scavenger FeTPPS decreased IR injury. GSNO also decreased the activation of AMP Kinase (AMPK) and its upstream kinase LKB1, both of which were activated in IR brain. AMPK has been implicated in nNOS activation via Ser1412 phosphorylation. To determine whether AMPK activation is deleterious in the acute phase of IR, we treated animals after IR with AICAR (an AMPK activator) and compound c (an AMPK inhibitor). While AICAR potentiated, compound c reduced the IR injury.ConclusionsTaken together, these results indicate an injurious nNOS/peroxynitrite/AMPK cycle following stroke, and GSNO treatment of IR inhibits this vicious cycle, resulting in neuroprotection and improved neurological function. GSNO is a natural component of the human body, and its exogenous administration to humans is not associated with any known side effects. Currently, the FDA-approved thrombolytic therapy suffers from a lack of neuronal protective activity. Because GSNO provides neuroprotection by ameliorating stroke’s initial and causative injuries, it is a candidate of translational value for stroke therapy.

GSNO promotes functional recovery in experimental TBI by stabilizing HIF-1α

HighlightsGSNO aids functional recovery in TBI animals by stimulating neurorepair.GSNO invokes neurorepair through S‐nitrosylation of HIF‐1&agr;.S‐nitrosylation stabilizes HIF‐&agr; and thus increases its activity.Inhibiting HIF‐1&agr; blocks GSNO‐mediated neurorepair mechanisms and functional recovery. ABSTRACT Traumatic brain injury (TBI) causes sustained disability due to compromised neurorepair mechanisms. Crucial to neurorepair and functional recovery following both TBI and stroke is hypoxia‐inducible factor‐1 alpha (HIF‐1&agr;). Based on reports that HIF‐1&agr; could be stabilized via S‐nitrosylation, we tested the hypothesis that the S‐nitrosylating agent S‐nitrosoglutathione (GSNO) would stabilize HIF‐1&agr;, thereby stimulating neurorepair mechanisms and aiding in functional recovery. TBI was induced by controlled cortical impact (CCI) in adult rats. GSNO (0.05 mg/kg) was administered at two hours after CCI. The treatment was repeated daily until the 14th day after CCI. Functional recovery was assessed by motor and cognitive functions, and the recovery was compared with the expression of HIF‐1&agr;. The mechanisms of GSNO‐mediated S‐nitrosylation of HIF‐1&agr; were determined using brain endothelial cells. While non‐treated TBI animals showed sustained neurobehavioral deficits, GSNO treatment of TBI improved neurobehavioral functions. GSNO also increased the expression of HIF‐1&agr; and VEGF. The beneficial effects of GSNO on neurobehavioral functions in TBI animals were blocked by treatment with the HIF‐1&agr; inhibitor 2‐methoxyestradiol (2‐ME). The stimulatory effect of GSNO on VEGF was reversed not only by 2‐ME but also by the denitrosylating agent dithiothreitol, confirming our hypothesis that GSNO’s benefits are mediated by the stabilization of HIF‐1&agr; via S‐nitrosylation. GSNO’s S‐nitrosylation of HIF‐1&agr; was further confirmed using a biotin switch assay in endothelial cells. The data provide evidence that GSNO treatment of TBI aids functional recovery through stabilizing HIF‐1&agr; via S‐nitrosylation. GSNO is a natural component of the human brain/body, and its exogenous administration has not shown adverse effects in humans. Therefore, the translational potential of GSNO therapy in TBI is high.

Oral administration of cytosolic PLA2 inhibitor arachidonyl trifluoromethyl ketone ameliorates cauda equina compression injury in rats

BackgroundPhospholipase A2 (PLA2)-derived proinflammatory lipid mediators such as prostaglandin E2 (PGE2), leukotrienes B4 (LTB4), lysophosphatidylcholine (LPC), and free fatty acids (FFA) are implicated in spinal cord injury (SCI) pathologies. Reducing the levels of these injurious bioactive lipid mediators is reported to ameliorate SCI. However, the specific role of the group IVA isoform of PLA2 cytosolic PLA2 (cPLA2) in lumbar spinal canal stenosis (LSS) due to cauda equina compression (CEC) injury is not clear. In this study, we investigated the role of cPLA2 in a rat model of CEC using a non-toxic cPLA2-preferential inhibitor, arachidonyl trifluoromethyl ketone (ATK).MethodsLSS was induced in adult female rats by CEC procedure using silicone blocks within the epidural spaces of L4 to L6 vertebrae. cPLA2 inhibitor ATK (7.5 mg/kg) was administered by oral gavage at 2 h following the CEC. cPLA2-derived injurious lipid mediators and the expression/activity of cPLA2, 5-lipoxygenase (5-LOX), and cyclooxygenase-2 (COX-2) were assessed. ATK-treated (CEC + ATK) were compared with vehicle-treated (CEC + VEH) animals in terms of myelin levels, pain threshold, and motor function.ResultsATK treatment of CEC animals reduced the phosphorylation of cPLA2 (pcPLA2) determined by Western blot, improved locomotor function evaluated by rotarod task, and reduced pain threshold evaluated by mechanical hyperalgesia method. Levels of FFA and LPC, along with PGE2 and LTB4, were reduced in CEC + ATK compared with CEC + VEH group. However, ATK treatment reduced neither the activity/expression of 5-LOX nor the expression of COX-2 in CEC + VEH animals. Increased cPLA2 activity in the spinal cord from CEC + VEH animals correlated well with decreased spinal cord as well as cauda equina fiber myelin levels, which were restored after ATK treatment.ConclusionThe data indicate that cPLA2 activity plays a significant role in tissue injury and pain after LSS. Reducing the levels of proinflammatory and tissue damaging eicosanoids and the deleterious lipid mediator LPC shows therapeutic potential. ATK inhibits cPLA2 activity, thereby decreasing the levels of injurious lipid mediators, reducing pain, improving functional deficits, and conferring protection against LSS injury. Thus, it shows potential for preclinical evaluation in LSS.

An NO/GSNO-based Neuroregeneration Strategy for Stroke Therapy

Stroke is associated with significant morbidity and mortality due to the limited neuroregeneration capacity of the injured brain. Other than thrombolysis in the acute phase of the disease by tissue plasminogen activator (tPA), which offers only a short window of treatment (~3 hours), an ideal stroke therapy is not available mainly because of limited understanding of the mechanisms of neuroregeneration and functional recovery in the chronic phase. Yet many drug therapies, including S-nitrosoglutathione (GSNO), have been shown to provide neuroprotection against acute disease in animal models of transient cerebral ischemia reperfusion (IR) and permanent ischemia. GSNO was also effective in stimulating neuroregeneration-related factors in the chronic phase of the disease. In this short review, we assess the evidence supporting exogenous administration of GSNO after experimental stroke as a means to stimulate neuroregeneration and aid in functional recovery via stabilization of the HIF-1α/VEGF pathway.

Combined treatment with GSNO and CAPE accelerates functional recovery via additive antioxidant activities in a mouse model of TBI

Traumatic brain injury (TBI) is the major cause of physical disability and emotional vulnerability. Treatment of TBI is lacking due to its multimechanistic etiology, including derailed mitochondrial and cellular energy metabolism. Previous studies from our laboratory show that an endogenous nitric oxide (NO) metabolite S‐nitrosoglutathione (GSNO) provides neuroprotection and improves neurobehavioral function via anti‐inflammatory and anti‐neurodegenerative mechanisms. To accelerate the rate and enhance the degree of recovery, we investigated combining GSNO with caffeic acid phenethyl ester (CAPE), a potent antioxidant compound, using a male mouse model of TBI, controlled cortical impact in mice. The combination therapy accelerated improvement of cognitive and depressive‐like behavior compared with GSNO or CAPE monotherapy. Separately, both GSNO and CAPE improved mitochondrial integrity/function and decreased oxidative damage; however, the combination therapy had greater effects on Drp1 and MnSOD. Additionally, while CAPE alone activated AMPK, this activation was heightened in combination with GSNO. CAPE treatment of normal animals also significantly increased the expression levels of pAMPK, pACC (activation of AMPK substrate ACC), and pLKB1 (activation of upstream to AMPK kinase LKB1), indicating that CAPE activates AMPK via LKB1. These results show that while GSNO and CAPE provide neuroprotection and improve functional recovery separately, the combination treatment invokes greater recovery by significantly improving mitochondrial functions and activating the AMPK enzyme. Both GSNO and CAPE are in human consumption without any known adverse effects; therefore, a combination therapy‐based multimechanistic approach is worthy of investigation in human TBI.