Reduced Nicotinamide Adenine Dinucleotide Phosphate, a Pentose Phosphate Pathway Product, Might Be a Novel Drug Candidate for Ischemic Stroke
Background and Purpose—Our previous study has defined a role of TP53-induced glycolysis and apoptosis regulator in neuroprotection against ischemic injury through increasing the flow of pentose phosphate pathway. We hypothesized that the pentose phosphate pathway product nicotinamide adenine dinucleotide phosphate (NADPH) could be a novel drug for treatment of ischemic stroke.
Methods—The NADPH was given before, at the onset, or after stroke onset with single or repeated intravenous (mice and rats) or intraperitoneal injections (monkey). The short- and long-term therapeutic effects of NADPH were evaluated in male adult ICR mice (total=614) with transient middle cerebral artery occlusion, in male adult Sprague–Dawley rats (total=114) with permanent middle cerebral artery occlusion, and in male adult rhesus monkey (total=12) with thrombotic middle cerebral artery occlusion.
Results—Administration of NADPH led to a dramatic increase in the levels of ATP and reduced form of glutathione, whereas it decreased the levels of reactive oxygen species. NADPH significantly reduced infarct volume, improved poststroke survival, and recovery of neurological functions in mouse and rat models of stroke. Robust neuroprotection of a single dose of NADPH was seen when it was administered within 5 hours after reperfusion; however, repeat administration of NADPH twice a day for 7 days starting 24 hours after the onset of stroke also offered therapeutic effects. Pretreatment with NADPH also significantly improved the outcome of stroke insult.
Conclusions—Administration of exogenous NADPH significantly protected neurons against ischemia/reperfusion-induced injury in 2 rodent stroke models. Thus, NADPH might be a promising drug candidate for treatment of ischemic stroke.
Ischemic stroke, the most common type of stroke, is the leading cause of death and disability in aged society,1 and the treatment options in clinic are limited. Thrombolytic therapy with recombinant tissue-type plasminogen activator is the first line therapeutic choice in stroke management, but its clinical application is limited because of the narrow therapeutic window and side effects.2 Less than 10% of patients with stroke will have the clinical indication for use of tissue-type plasminogen activator. Therefore, developing novel therapeutic drugs for ischemic stroke is urgently needed.
The interruption of blood flow to brain tissue during stroke attack causes a metabolic crisis, which results in reduced energy supply and a burst increase in reactive oxygen species (ROS). It is generally accepted that neurons are particularly vulnerable to ischemic injury because neurons have little activity of anaerobic glycolysis. Meanwhile, neurons consume a large amount of energy for maintaining normal neurotransmission. Many studies have indicated that oxidative stress is a key pathological player in ischemic brain injury and have proposed it as a therapeutic target.3–5 However, previous studies with various antioxidants offered limited clinical efficacy in patients with stroke.3 One possible reason is that ROS are produced inside of cells and before they are released into extracellular space, the oxidative damage of DNA, proteins and lipids have already been produced. Thus, scavenge of extracellular space ROS may have limited benefits to neurons.
TP53-induced glycolysis and apoptosis regulator was reported to inhibit glycolysis and increase the flow of pentose phosphate pathway.6 It plays an important survival role in cancer cells.6 Our previous studies found that TP53-induced glycolysis and apoptosis regulator was predominantly expressed in brain neurons and rapidly responded to ischemia/reperfusion insult. TP53-induced glycolysis and apoptosis regulator exerted neuroprotection against ischemic brain injury through increasing the flux of pentose phosphate pathway.7 Nicotinamide adenine dinucleotide phosphate (NADPH), a metabolic product of pentose phosphate pathway, is a coenzyme and classic molecule involved in many anabolic reactions in cells.8 It serves as hydrogen and electron donors in reductive biosynthesis of amino acids, lipids, and nucleotides. Another important biological function of NADPH is to provide redox power to antioxidant systems that mediate the cell response to oxidative stress.9–11 Reduced form of glutathione is essential for the functions of key antioxidant enzyme glutathione peroxidase,12 and NADPH is required for its regeneration. NADPH is also implicated in the production of ATP, and together with the respiratory chain-oxidized NAD, it maintains cell energy homeostasis.9,13 We expected that supply of exogenous of NADPH would reduce intracellular oxidative stress and improve energy supply after stroke attack. This study, thus, was sought to investigate whether supplementation of exogenous NADPH had a therapeutic effect in rodent and primate models of ischemic stroke. The results of this study strongly suggest that NADPH may be a novel therapeutic drug for stroke.
Materials and Methods
Middle Cerebral Artery Occlusion Model in Rodents
Male ICR mice (25–30 g) and male adult Sprague–Dawley rats (250–280 g) were purchased from SLACCAL Lab Animal Ltd (Shanghai, China). All animals were used in accordance with the institutional guidelines for animal use and care, and the study protocol was approved by the Ethical Committee of Soochow University. Middle cerebral artery occlusion (MCAO) surgery was performed as described previously.14,15 In brief, mice and rats were anesthetized with intraperitoneal injection of 1% pentobarbital sodium, the right external carotid artery was exposed, and a small incision was made. A 6-0 (for mouse) or 3-0 (for rat) monofilament nylon suture (Doccol Co, Redlands, CA) was inserted from the external carotid artery into the internal carotid artery to occlude the right MCA at its origin. Two hours after occlusion, filament was withdrawn to allow blood reperfusion. Sham-operated animals underwent identical procedures, except for the suture insertion. Cerebral blood flow was monitored (LDF, ML191 Laser Doppler Blood Flow Meter, Australia), and only those animals with 90% reduction of blood flow during MCAO and 85% to 95% recovery of blood flow during reperfusion were used (Figure I in the online-only Data Supplement). A homoeothermic heating blanket was used to maintain the core body temperature at 37°C during ischemia/reperfusion operation. For permanent MCAO (pMCAO), the occluding filament was left in situ until the end of experiment. Mice and rats were euthanized post ischemia/reperfusion at indicated time. Brains were harvested and stored at −80°C for further analysis.
In the experiments using rhesus monkeys (age, 6–8 years), animals were allocated to experimental groups, so that the differences in the average body weight among groups were minimized. In the experiments using mice and rats, whose body weights were relatively uniform, the animals were randomly allocated to each experimental group. The criteria for exclusion, the results of the exclusion, and mortality in each group are shown in Table I in the online-only Data Supplement. All of the evaluations and administration of drug in the experiments were performed in a masked manner.
Measurement of Infarct Volume in Rodents
The mice and rats were anesthetized with 1% pentobarbital sodium, and brains were rapidly removed 24 hours or 7 days after MCAO. Brains were sliced with plastic modules (Harvard Apparatus, MA), and sections were incubated in 1% 2,3,5-triphenyltetrazolium chloride in saline for 20 minutes at 37°C. The infarct area was measured on 5 sections (mice, 2-mm thickness; rat, 3-mm thickness) per brain using digital imaging and image analysis software (Image Pro Plus, Media Cybernetics, Silver Spring, MD) by an observer who was blinded to experimental conditions. The presence or absence of infarction was determined by examining 2,3,5-triphenyltetrazolium chloride–stained sections for the areas on the side of infarction that did not stain with 2,3,5-triphenyltetrazolium chloride. Infarct volume was expressed as a percentage of total hemisphere.14,16
Measurement of Neurological Score and Brain Water Content After MCAO
Behavioral deficits were evaluated 24 hours after reperfusion by an observer blinded to the condition of treatments according to the method previously described.17 The following rating scores were used: score 0, no neurological deficit; score 1, failure to extend left forepaw fully; score 2, circling to the left; score 3, falling to the left; and score 4, did not walk spontaneously and had a depressed level of consciousness. Mice and rats that did not show behavioral deficits immediately after reperfusion (neurological score, 0) were excluded from the study.
After the wet weight of the brains was quantified, the red and white parts of these brains were desiccated at 105°C for 48 hours until the weight was constant. The total weight of the dried 2,3,5-triphenyltetrazolium chloride–stained brains was obtained, and the water content of each brain was measured as follows: water content=(wet weight−dried weight)/wet weight×100%.18
Evaluation of the Blood–Brain Barrier Integrity
The integrity of the blood–brain barrier was evaluated by using Evans blue extravasations. Evans blue (2% in saline, 4 mL/kg) was injected intravenously at 22 hours after reperfusion. The sample collection and measure protocol were followed as the previous study.19
Thromboembolic MCAO Model in Monkeys
Twelve male adult rhesus monkeys (age, 6–8 years; weight, 6–8 kg) were purchased from Yongfu Laboratory Animal Breeding Base (Guilin, Guangxi, China). Twelve monkeys were randomly divided into 2 groups: NADPH (N7505; Sigma, Saint Louis, MO) group and saline group, 6 in each group. All animals were fasted for 24 hours with free access to water before surgery. Monkeys were intramuscularly injected with composite anesthetic (ketamine 10 mg/kg+midazolam 1 mg/kg+scopolamine 0.02 mg/kg). Anesthetized animals were examined with magnetic resonance imaging (MRI) T2-weighted scanning before surgery to exclude intracranial lesions. After MRI scanning, all animals were sent to interventional radiological operation room with nasal oxygen, ECG, respiration, and blood pressure monitor systems. Intravenous injection of propofol was used to maintain anesthesia. Animal autologous blood (5 mL) with heparin was centrifuged for 3 minutes, and the upper layer serum was collected. Serum was added with coagulase and injected into an epidural anesthesia tube (1 mm diameter; Henan Camel Medical Device Company, Xinxiang City, China) to make blood clots. The femoral artery was punctured using the Seldinger technique, and the microcatheter was introduced to the compartment M2 segment of MCA through a 4F guiding catheter (all catheter and guidewire were from Cordis corporation production [New York]). The blood flow was monitored with MCA angiography. The 3-cm length of blood clot was injected into MCA through the microcatheter. The occlusion of blood flow was confirmed with angiography. After 30 minutes of MCAO, the NADPH group was administered 1 mg/kg of NADPH by intraperitoneal injection. All monkey procedures were approved by the Ethical Committee of Guilin Medical College Affiliated Hospital.
Measurement of Infarct in Monkeys With MRI
MRI was performed in all animals with 3T (Magnetom Verio; Siemens Medical Systems) equipped with a 32-channel system, axial slice scanning. MRI sequences and parameters are detailed in Table I in the online-only Data Supplement. Both T2WI and diffusion-weighted imaging were taken 2 hours after MCAO. Animals were then awakened and allowed to recover. All animals were reanesthetized and scanned 24 hours and 10 days after MCAO. The ischemic brain tissue also showed a high signal in diffusion-weighted imaging, but the infarct area in T2WI was seen clearer than that in diffusion-weighted imaging because of the brighter pixels; so we defined the infarct area using T2WI images. All images were amplified to outline the boundary of infarction and the infarct area then be measured using a MRI workstation.
Measurement of NADPH Levels
Mice were intravenously administered with NADPH (2.5 mg/kg). Mice (6 per group) were anesthetized with 1% pentobarbital sodium and decapitated. Brains were removed and immediately dissected on a cooled ice pack (−20°C) at the indicated time. Brain and blood NADPH concentrations were measured at the indicated time with the enzychrom NADPH/NADP assay kit (BioAssay Systems, Hayward, CA) following manufacturer’s instructions.
Measurement of ATP Levels
Mice were intravenously administered with NADPH (7.5 mg/kg) at the onset of reperfusion. Mice (6 per group) were anesthetized with 1% pentobarbital sodium and decapitated 3 hours after MCAO/reperfusion. Brains were removed and immediately dissected on a cooled ice pack (−20°C). In each hemisphere, the whole-ischemic cortex (infarct tissue and penumbra tissue) was collected and quick frozen in liquid nitrogen. The ATP content in cortex was measured at 3 hours after reperfusion with the ATP assay kit (Beyotime, Nantong, China) following manufacturer’s instructions.
Measurement of ROS
For assessing ROS in vivo, dihydroethidium (2 mg/kg, Sigma) was injected intraperitoneally 1 hour before the end of experiment. Mice were anesthetized and decapitated, and brain sections (10 μm) were cut with a cryostat (Leica). The dihydroethidium fluorescence in brain sections was observed with a fluorescence microscope (Olympus, Tokyo, Japan).
Data were presented as mean±SEM and were evaluated with 1-way ANOVA with Bonferroni multiple comparisons post hoc test for comparisons of >2 means and unpaired Student t test if the statistical differences between 2 groups were compared. Differences in survival rates were assessed using the Log-rank test followed by Holm–Sidak method for all pair-wise multiple comparisons. The multiple comparison methods are described in the figure legends. A difference was considered statistically significant when P<0.05.
Exogenous NADPH Performs Biological Functions
To test whether exogenous NADPH can get into mouse brain tissues and neurons, normal mice were given intravenous injection of NADPH of 2.5 mg/kg and the concentrations of NADPH in plasma and brain were determined. The results showed that the injection of NADPH significantly increased NADPH levels in mouse blood and brain tissues. The half-life of NADPH was ≈6 hours in mouse blood (Figure 1A) and 7 hours in brain tissues (Figure 1B). Next, we determined whether exogenous NADPH produced biological functions under ischemia and reperfusion conditions. The supply of NADPH did not elevate ATP levels under normal conditions. However, ATP levels in mouse brain were markedly decreased after ischemic attack. Intravenous administration of NADPH significantly increased brain ATP levels during reperfusion period (Figure 1C). Dihydroethidium staining is a reliable method for detecting ROS production.20 NADPH administration significantly reduced the number of dihydroethidium-positive cells and intensity of dihydroethidium staining in the ischemic brain sections (Figure 1D). Moreover, the levels of NADPH and reduced form of glutathione in brains of control mice were significantly decreased during reperfusion but were reversed by the NADPH administration (Figure IIA and IIB in the online-only Data Supplement). To further confirm whether the exogenous NADPH penetrates into brain tissue through the blood–brain barrier, the level of NADPH was determined in mice brains with or without perfusion (to remove blood in brain). The results demonstrated that NADPH levels were significantly increased by injection of exogenous NADPH in the brain tissue with or without perfusion, suggesting that exogenous NADPH may, at least partially, penetrate into brain tissues (Figure IIC in the online-only Data Supplement).
To determine whether NADPH penetrates into the plasma membrane of neurons, NADPH was added to cultured mouse cortical neurons and intracellular NADPH was measured. The results showed that intracellular NADPH concentration in cultured mouse cortical neurons was significantly increased 2 hours after the addition of exogenous NADPH (Figure IID in the online-only Data Supplement). To further demonstrate whether the biological actions of exogenous NADPH were produced in the neurons, cortical primary neurons were exposed to oxygen–glucose deprivation/reoxygenation and the effects of NADPH on neuronal bioenergy homeostasis were determined. The results showed that NADPH did not change the ATP level in neurons under normal conditions. However, NADPH induced a drastic increase in the ATP level in the neurons 3 hours after oxygen–glucose deprivation/reoxygenation (Figure IIE in the online-only Data Supplement). The results also indicated that NADPH had no effect on neuronal survival as measured 3 hours after reoxygenation and dead cells did not produce ATP (Figure IIE and IIF in the online-only Data Supplement). These data suggest that NADPH can increase the production of ATP in survival neurons when oxygen becomes available again. There was a significant decrease in reduced form of glutathione levels in the cortical neurons after oxygen–glucose deprivation/reoxygenation when compared with control, whereas higher levels of reduced form of glutathione were observed in the neurons that were treated with NADPH (Figure IIG in the online-only Data Supplement). Moreover, the ROS levels in primary neurons (dihydroethidium-positive cells) were significantly increased to ≈6-folds when compared with their control group. The treatment of neurons with NADPH reduced ROS levels (Figure IIH and III in the online-only Data Supplement). These studies confirmed that exogenous NADPH could get into neurons to perform its biological functions.
To test whether NADPH has other novel biological actions, we determined the in vivo and in vitro effects of NADPH on platelet aggregation. We found that pretreatment with NADPH for 30 minutes in rats dramatically suppressed the ADP-induced platelet aggregation (Figure IIIA and IIIB in the online-only Data Supplement). Meanwhile, we demonstrated that in vitro administration of NADPH (30–90 μmol/L) also inhibited ADP-induced platelet aggregation in a dose-dependent manner (Figure IIIC and IIID in the online-only Data Supplement). In addition, we also found that treatment with NADPH 30 minutes after pMCAO mildly increased cerebral blood flow in ischemic hemisphere (Figure IIIE and IIIF in the online-only Data Supplement).
Exogenous NADPH Reduces Ischemic Injury in 2 Rodent Stroke Models
We then asked whether exogenous NADPH could be a therapeutic agent for stroke treatment. We used a mouse brain ischemia/reperfusion model to determine the dose–response of the exogenous NADPH on brain ischemic injury. Mice were intravenously administered with NADPH 2.5, 5, and 7.5, mg/kg (immediately at reperfusion), and infarct volume and neurological deficits were assessed 24 hours after ischemic insult. The data showed that NADPH reduced infarct volume from 59.99±2.77% to 39.61±4.03%, 38.36±3.69%, and 30.41±4.10%, respectively (P<0.05 and P<0.01 compared with vehicle [model] mice; Figure 2A and 2B). The administration of NADPH also markedly attenuated ischemia/reperfusion-induced behavioral deficits and increases in water content (P<0.05 and P<0.01 compared with vehicle mice; Figure IVA and IVB in the online-only Data Supplement). Because 7.5 mg/kg of NADPH displayed more powerful neuroprotective effects, we next determined the therapeutic windows of a single dose of NADPH on ischemic brain injury with this dose. The results showed that intravenous administration of a single dose of NADPH within a 5-hour time window after reperfusion was effective (P<0.05 and P<0.01; Figure 2C and 2D; Figure IVC and IVD in the online-only Data Supplement). Furthermore, a single dose (administered at the onset of reperfusion) or multiple doses (administered 0, 4, and 8 hours after reperfusion) of NADPH produced similar effects on reduction in infarct volume, behavioral deficits, and water content (Figure 3A–3C). No significant difference between the 2 dose regimes was observed in the recovery of motor and cognitive functions (Figure VA–VC in the online-only Data Supplement) but seemed to increase the long-term survival rate 28 days post ischemia/reperfusion with multiple doses of NADPH (treated with NADPH at the onset and 8 hours after reperfusion and then twice a day for 3 weeks; Figure 3D). To characterize the effects of NADPH on postischemic brain pathology, we examined global morphology and histology of mouse brain 28 days post ischemia/reperfusion. Both single and multiple doses of NADPH robustly alleviated the shrinkage of the ischemic hemisphere (P<0.01; Figure VIA and VIB in the online-only Data Supplement). Meanwhile, profound neuronal loss was observed in control brains, but NADPH treatment markedly increased the number of surviving neurons in ischemic cortex (Figure VIC in the online-only Data Supplement).
To further confirm the protective effect, we evaluated the efficacy of NADPH in mice and rats using filament-induced pMCAO, a procedure in which no complete blood reperfusion occurs.21 Administration of NADPH 1 hour after pMCAO also decreased infarct volume to 34.81±4.83% from 59.08±2.11% (P<0.01; Figure 4A), behavioral score to 1.93±0.15 from 2.87±0.24 (P<0.01; Figure 4B), and brain water content from 83.72±0.45% to 81.42±0.68% (P<0.01) 24 hours after pMCAO (Figure VIIA in the online-only Data Supplement). Similar results were obtained in rats with pMCAO, as shown by the data that NADPH administration reduced infarct volume from 57.78±1.57% to 30.09±6.78% (P<0.01; Figure 4C), neurological score from 3.2±0.19 to 1.9±0.23 (P<0.01; Figure 4D), and water content from 82.52±0.77% to 79.85±0.48% (P<0.05; Figure VIIB in the online-only Data Supplement). In addition, the effects of multiple doses of NADPH were also evaluated in rats that received NADPH at 2 and 8 hours after pMCAO and then twice a day until 7 days of postischemic survival. Cresyl violet staining (Figure VIIC in the online-only Data Supplement) was used to determine the neuroprotective effects of the NADPH 7 days after the induction of ischemia. The sham group showed normal appearance of the neuronal nuclei, whereas dead cells in the ischemic cortex (treated with vehicle) displayed the pyknotic nuclei. The administration of NADPH dramatically increased survival neurons in ischemic cortex (Figure VIID in the online-only Data Supplement). Prolonged treatment with NADPH also significantly alleviated pMCAO-induced neurological deficits (P<0.05 and P<0.01; Figure VIIE in the online-only Data Supplement). All rats that received multiple doses of NADPH survived, whereas only 6 rats of 12 in control survived 7 days after pMCAO (P<0.05; Figure VIIF in the online-only Data Supplement).
Preliminary Observations of the Effects of Exogenous NADPH in a Primate Stroke Model
To test the therapeutic potential of NADPH in primates, we evaluated the neuroprotective effects of NADPH in rhesus monkeys with thrombotic occlusion of M3 segment of brain MCA.22 Representative MRI scanning images showed that NADPH administration (1 mg/kg) reduced ischemia–induced infarct areas (Figure 5A). Quantification of serial brain images revealed that NADPH significantly reduced the infarct volume from 5.53±0.94 mm3 to 2.69±0.84 mm3 24 hours after ischemic onset (P<0.05; Figure 5B). To examine the long-term benefit, we quantified infarction areas and found that NADPH administration significantly reduced infarction areas in the cortex 10 days post ischemia (Figure 5C). Higher clinical examination rating scores were observed in saline-treated monkeys at 24 hours after ischemia, which was ameliorated in NADPH-treated monkeys (P<0.01; Figure 5D). The amelioration of rating scores by single dose of NADPH persisted for 29 days after ischemic attack (Figure VIII in the online-only Data Supplement).
Prestroke or Poststroke Treatment With Exogenous NADPH Produces Robust Beneficial Effects
To determine whether preventive treatment with exogenous NADPH had a beneficial effect on the outcome of ischemic stroke, mice were intravenously administered with 7.5 mg/kg of NADPH twice a day for 7 days. Mice were subjected to ischemia/reperfusion insult and infarct volume, and neurological deficits were assessed 24 hours after ischemic insult. No NADPH was given during and post stroke. The result showed that ischemia/reperfusion-induced infarct volume and neurological deficits were markedly reduced by pretreatment with NADPH (Figure 6A and 6B). The Evans blue extravasation and brain water content were significantly induced 24 hours post ischemia/reperfusion by pretreatment with NADPH (Figure 6C; Figure IXA in the online-only Data Supplement). Furthermore, we examined whether continuous administration of NADPH after stroke offers beneficial effects on recovery of neurological functions. The NADPH (7.5 mg/kg) was given twice daily for 14 days starting 24 hours after stroke insult. The results showed that NADPH significantly increased the long-term survival rate and the recovery of motor and cognitive functions and 28 days post ischemia/reperfusion (Figure IXB–IXE in the online-only Data Supplement). The morphological examination demonstrated that exogenous NADPH notably decreased the shrinkage of the ischemic hemisphere (Figure 6D).
This study demonstrated, for the first time, that systemic administration of exogenous NADPH significantly protected neurons against ischemia/reperfusion-induced injury in 2 rodent stroke models. It not only substantially reduced infarct volume after stroke insult but also significantly reduced long-term mortality and improved recovery of neurological functions. The extent of neuroprotection offered by NADPH is exceptional, and the therapeutic window of NADPH efficacy is much larger than that of tissue-type plasminogen activator23 in rodent models. NADPH could also be used for preventive treatment to reduce ischemic injury and poststroke treatment to improve survival of animals and recovery of neurological functions in rodent models. The acute toxicity of NADPH seemed to be low as mice could tolerate approximately a 400-fold increase of its therapeutic dose (Mice were slowly injected with NADPH [1, 2 or 3 g/kg] via tail vein. No animal died after the drug administration. Twenty mice were used in this preliminary experiment). Therefore, we propose that NADPH may be a novel drug candidate for stroke therapy. This notion is strongly supported by several unique features of NADPH and its biological functions.
Effective therapeutic agents for the treatment of stroke must enter into the brain parenchyma through the blood–brain barrier, which is one of the major obstacles for treating central nervous system diseases.24 This study demonstrated that NADPH levels in brains were increased after intravenous injection of NADPH. This study further demonstrated that addition of NADPH in culture medium significantly increased intracellular NADPH. Furthermore, administration of exogenous NADPH increased the levels of reduced form glutathione and ATP, suggesting that NADPH can effectively penetrate the blood–brain barrier and cell membranes to produce biological functions. It should be pointed out that the brain NADPH we measured in this study might only reflect NADPH accumulation in the vasculature but not in the brain cells.
One of the functions of NADPH is to produce reduced form of glutathione. This property enables exogenous NADPH to block the elevation of intracellular ROS levels during ischemia/reperfusion. In addition, when oxygen is available, excess NADPH can be used by mitochondria for production of ATP.9 A rapid recovery of ATP levels after ischemic insult certainly favors rescue of neurons to avoid irreversible injury. In an attempt to find novel biological actions of NADPH, we evaluated whether NADPH inhibits platelet aggregation. A complete blockade of cerebral blood flow in our MCAO and pMCAO models was reached because of the formation of blood clot around the head of inserted nylon suture. Thus, inhibition of platelet aggregation may suggest that NADPH could improve blood supply to ischemic hemisphere. In fact, a slight increase in blood flow to ischemic cortex was observed in a pMCAO model. Although these observations remain to be carefully evaluated in a well-designed study in future, we believe that the inhibitory effect on platelet function is an additional benefit of NADPH for stroke treatment.
One of the big challenges in stroke management is missing the optimal therapeutic window when patients administered into hospital. This study demonstrated that a single dose of NADPH remained effective if administered within 5 hours after onset of reperfusion. However, NADPH still produced therapeutic benefits when administered 24 hours after onset of stroke if multiple dosages were used for prolonged time period. To test the rationale for preventive use of NADPH, we administered NADPH twice a day for 1 week and stopped the treatment on the day of onset of stroke and thereafter. The results demonstrated that preventive treatment with NADPH robustly reduced ischemic injury and improved long-term survival and recovery of neurological functions. Thus, NADPH could be a drug for acute treatment, preventive treatment, and poststroke treatment.
As no single animal model can completely mimic the pathology of human ischemic stroke, multiple animal models were used for evaluation of short- and long-term therapeutic effects of NADPH in the present study. The short- and long-term therapeutic effects of NADPH were verified in multiple animal stroke models. As people may argue that in human patients with stroke, complete reperfusion of blood may not occur; thus, this study tested the protective effects of NADPH in mouse and rat models of pMCAO, in which blood flow was permanently interrupted. The results demonstrated that NADPH reduced infarct volume, increased long-term survival, and recovery of neurological functions. Considering the fundamental differences between rodent brains and human brains, we also evaluated the therapeutic effects of NADPH in a primate stroke model, which represents the best model for human ischemic stroke.22 In this model, focal blood flow was blocked by direct injection of a thrombus into the midbrain artery. This procedure produced brain infarct in the territory supplied by M3 segment of middle brain cerebral artery. The initial studies showed that NADPH was effective as evidenced by reductions in infarct volume and neurological deficits. However, only limited number of monkeys was used in this study, and the infarct area was small with current protocol. The efficacy of NADPH in primates remains to be further confirmed.
It should also be pointed out that there are additional flaws in this study. The selection of dosage of NADPH in mice, rats, and monkeys was not carefully converted according to pharmacokinetics in different species in this study. The routes of NADPH administration in rodents and monkeys were also different. These pitfalls might affect the reliability of the study. Furthermore, except for antioxidative stress offered by NADPH, the other mechanisms that may also be responsible for NADPH’s robust neuroprotective effects remain to be further determined.
This study showed that NADPH offered robust short- and long-term therapeutic effects in 2 rodent stroke models. NADPH may have clinical advantages as it has a relatively big therapeutic window, low acute toxicity, and no risk of cerebral breeding. NADPH had neither beneficial nor harmful effect on a hemorrhage stroke model (our unpublished observations). The biological actions of NADPH also strongly suggest that NADPH may prolong tissue-type plasminogen activator’s therapeutic window and prevent secondary injury associated with reperfusion after thrombolysis. Thus, NADPH might be a promising drug candidate for therapeutic intervention of cerebral ischemia.
Sources of Funding
This study was supported by the Natural Science foundation of China (no. 31500822, 81271459, 31030034), the “973” project from the Ministry of Science and Technology of China (2011CB51000), the Priority Academic Program Development of Jiangsu Higher Education Institutes, and the Graduate Education Innovation Project of Jiangsu Province (CXZZ12_0850).
↵* Drs Li and Zhou contributed equally.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.115.009687/-/DC1.
- Received May 13, 2015.
- Revision received October 11, 2015.
- Accepted October 16, 2015.
- © 2015 American Heart Association, Inc.
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