1. Introduction
Aging and neurodegeneration due to damage by free radicals (FRs) are very well-known biological events at the origin of stroke and neurodegenerative diseases (NDS) such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and cancer, to mention some of the most devastating examples [
1].
There is scientific evidence to support the relationship between stroke, or NDs, and the excessive production of reactive oxygen species (ROS) [
2]. An accumulation of oxidative damage (OD) may contribute to the delayed onset and progressive nature of NDs [
3]. One important factor contributing to neurodegeneration is that the central nervous system is unable to cope sufficiently with excessive FRs [
2]. Thus, the neuroprotection strategy is considered one of the most attractive options to avoid FRs’ toxic effects [
4]. Dementia due to cerebral ischemic lesions is relatively common in older people. In other words, multiple (micro)infarcts may cause dementia [
5,
6].
The ‘FR hypothesis of aging’ maintains that: (1) endogenous antioxidant defense mechanisms are insufficient to detoxify all oxygen FRs continually being generated, and (2) resulting OD to critical biological molecules, such as DNA, protein, and membrane lipids, contributes to age-related neuronal loss and/or dysfunction [
7]. Supportive of this hypothesis are the studies showing increased OD during normal brain aging in AD and PD [
8]. Consequently, ROS are important causative factors in normal brain aging and NDs, and chronic enhancement of antioxidant defenses should slow this process, resulting in improved cognitive and/or motor function.
On the other hand, excitotoxicity has been linked to oxidative stress (OS).
N-methyl-D-aspartate (NMDA) has been shown to increase FR generation in vivo [
9]. FR spin traps exert neuroprotective effects against both glutamate and NMDA toxicity in vitro [
10]. It has been suggested that excitotoxicity and OS may be sequential and share interactive mechanisms, leading to neuronal degeneration [
11,
12].
Stroke is a condition affecting an increasing number of people worldwide and is the main cause of disability [
13]. The ischemic cascade begins with energy failure due to the obstruction of a blood vessel that produces a massive and prolonged release of glutamate [
14]. Physiopathological events associated with brain ischemia are related to OS process, Ca
2+ dyshomeostasis, mitochondrial dysfunction, pro-inflammatory mediators, and/or programmed neuronal cell death [
15]. In the ischemic stroke, as the result of the obstruction of a blood vessel, a critical reduction of oxygen–glucose supply and cerebral blood flow (less than 25%) occur in brain [
16]. Thus, under deprivation of oxygen and glucose, cell death takes place in two phases: cell death from anoxia/hypoxia and energy depletion [
17], followed by reperfusion that increases OS and FR formation, excitotoxicity, and nitric oxide (NO) production with ulterior energy failure and delayed death [
18].
No effective therapeutic drugs to treat or prevent brain damage in ischemic stroke are available. Currently, the therapy for acute ischemic stroke has two basic principles: (1) dissolving the intravascular occlusion (
reperfusion), which may refer to reperfusion injury (RI) (tissue damage caused when blood supply returns to the tissue) or reperfusion therapy (the medical treatment that restores blood flow through blocked arteries, typically after a ichemic attack), and (2) preserving the brain from the harmful cellular and metabolic cascade (
neuroprotection). It is known that ischemia with RI leads to an enhanced production of FRs and that this production contributes to OD [
19]. Even if ischemia is not followed by RI, significant amounts of ROS may be generated in brain tissue with some degree of residual perfusion, as demonstrated in the ischemic penumbra during occlusion of the middle cerebral artery (MCAO) in rats.
Nowadays, the only treatment approved for stroke is the recombinant tissue plasminogen activator (rtPA). rtPA is used to open a blood vessel, preventing brain damage at the ischemic penumbra, which, despite being a hypoperfused and non-functional tissue, is a viable tissue adjacent to the infarcted core. However, rtPA has a very narrow therapeutic window (3.5 h) [
20].
Thus, in the search for alternative therapies to rtPa [
21] to prevent its secondary effects and improve its narrow therapeutic window, new therapeutic agents are needed to recover tissue functionality before cell death and to be effective against several targets, including excitotoxicity and disturbed Ca
+2 homeostasis [
22], mitochondrial failure [
23], OS and nitrosative stress [
24], inflammation [
25], and apoptosis [
26].
As part of this effort, two small molecules, edaravone [
27] (
Figure 1) and 3-
n-butylphthalide [
28] (
Figure 1) have been recently approved for the therapy of stroke in Japan and China, respectively.
In addition, and in this context, nitrones [
29,
30] (I,
Scheme 1) have been largely investigated as targets of choice. Nitrones are small spin-trapping organic compounds that readily react with a variety of FRs, forming unreactive, more stable spin-adducts, a property that was first used to detect FRs by electron paramagnetic resonance. There is significant evidence supporting the notion that FR production is a key factor in the development of brain injury following both cerebral ischemia (CI) and RI. The use of compounds which trap FRs to ameliorate ischemic damage is, therefore, a logical therapeutic approach. Thus, not surprisingly, nitrones occupy a privileged place for stroke and other human conditions, such as NDs [
31], diseases of aging [
32], cancer [
33], cardiovascular disease [
34], renal injury [
35], visual loss [
36], and acoustic trauma [
37,
38].
Nitrones are compounds that are able to trap ROS and reactive nitrogen species (RNS), such as
●OH, O
2●−, NO
●, and ONO
2−, and non-radical molecules [hypochlorous acid, or hydrogen peroxide (H
2O
2)] [
39], as common biological events involved in the progress of diseases linked to OS. Nitrones are potent antioxidant molecules [
40] that are able to reduce OS. Nitrones’ power to scavenge the different types of ROS derives from their activated carbon–nitrogen double bond [R
1R
2C=N
+(O
−)R
3 (I)] that prompts the easy reactive species [R]
● nucleophilic radical attack, leading to the less reactive and harmful nitroxide II species (
Scheme 1) for biological targets, resulting in cell survival, tissue, biomolecules, and membrane stability [
41]. However, recent results, such as the fact that the doses used for spin-trapping experiments are 1000-fold higher than those usually applied in the in vitro neuroprotection analyses (10–50 μM), and that the amounts of nitrones in vivo are currently under 50 μM (clearly insufficient to trap ROS/RNS), suggest that other mechanisms are responsible for nitrones’ scavenging capacity [
39]. Nowadays, it is widely accepted that nitrones may suppress signal transduction processes, affording significant anti-inflammatory/anti-apoptotic [
42] and NO-releasing properties [
43] (these events being, most possibly, the origin of their antioxidant/neuroprotective profile [
39]).
The literature is full of articles and reviews that have covered the organic [
44], physicochemical/computational [
45], and biological aspects of these molecules [
46]. In addition, collateral topics, such as “antioxidants” [
47], “ROS”, and “spin-trapping agents” [
48], usually include nitrones in their accounts, resulting in an impressive mass of information underlying their medicinal chemical features [
49].
Based on the understanding of the biochemical processes involved in the formation and development of a stroke, a number of products have been developed that target the different ischemic and RI events. Despite the promising initial results, neuroprotection drugs for stroke have failed in advanced clinical phases; consequently, no neuroprotective agent has been approved by the Food and Drug Administration (US) for stroke therapy.
However, neuroprotection is still a choice, and OS is a suitable biological target. In this context, antioxidants such as α-phenyl-
N-tert-butylnitrone (PBN) (
Figure 1) [
50], NXY-059 (
Figure 1) [
51], and TBN [
52] (
Figure 1) have been developed, resulting in therapeutic candidates for cancer [
53], NDs [
54], hearing loss [
42], and stroke [
55].
NXY-059 (
Figure 1) [
56] is a well-known FR scavenger with a good neuroprotective profile in rat models of transient/permanent focal ischemia and in rodent models of stroke. It has been launched several times in advanced clinical studies without success [
57]. In spite of this,
tert-butylnitrones, such as NXY-059, are known to afford
tert-butylhydroxylamines after hydrolysis as powerful radical scavengers that, further, could be oxidized to 2-methyl-2-nitrosopropane, which may then produce NO, the source and origin of the neuroprotection, as has already been reported for NO donor molecules [
58].
On the other hand, recent reports have highlighted the powerful neuroprotective effect shown by new PBN derivatives bearing
N-aryl substituents on human neuroblastoma cells under induced in vitro experimental OS [
59]. In this study, we will focus on the neuroprotection power and capacities attributable to PBN and PBN-derived nitrones [
39], the recent advances to prevent neuron death after stroke and RI [
60]. This means, obviously, a selection of nitrones that we have restricted to PBN-analogues [
39] (
Figure 1), developed in the author’s laboratory. Other PBN-nitrones developed in other groups have already been reviewed [
30,
39] and are not going to be discussed here.
2. PBN, an Antioxidant and Neuroprotective Agent
PBN (
Figure 1) is one of the best-known and investigated spin traps of ROS/RNS. PBN is actually a better scavenger for non-lipid radicals (such as hydroxyl, for instance) than for lipid radicals such as peroxyl and alkoxyl. PBN is both hydrophilic and lipophilic; it readily permeates all tissue, including brain tissue; its half-life in blood is 3–4 h [
8]; and its concentration in plasma has been shown to peak at 15 min, while the maximum in other organs tested occurred at 30 min. When administered intraperitoneally (IP) to rats, the amount of PBN per gram of tissue is always higher in the liver and kidney than in the brain [
61].
When injected into older adult gerbils, PBN led to the removal of oxidative damaged proteins, recovery of the ability to perform in a spatial radial maze test, reversal of age-related loss in the stimulation of dopamine release [
62], affects on physiological functions, and extended life span in treated animals [
63,
64]. PBN releases NO after the reaction with ROS in vitro, and this release may play a key role in these activities.
Accumulating evidence has implicated ROS production and the resultant OD as a major contributing factor in brain aging and cognitive decline. It was shown [
8] that chronic PBN treatment of 24-month-old rats for up to 9.5 months improved cognitive performance in several tasks, resulting in greater survival during the treatment period and decreased OD within brain areas important for cognitive function. These results not only provide a direct link between FRs/OD and cognitive performance in old age, but also suggest that antioxidants could be developed to treat or prevent age-related cognitive impairment and AD.
In 1990, in a preliminary communication [
65], and in 1991, in a full paper [
66], Phillis and Clough-Helfman reported that the IP administration of PBN (100 mg/kg) 30 min prior to transient MCAO bilateral 5 min (forebrain ischemia) in gerbils prevented the increase in locomotor activity observed in saline-injected ischemic animals and significantly reduced OD to the hippocampal cornu ammonis 1 (CA1) pyramidal cell layer observed 5 d post-ischemia. Measurements of body temperature revealed that the administration of PBN and the induction of CI were associated with small reductions in body temperature, but these changes were not significant. Finally, it was observed that PBN (100 mg/kg) administered 2 h post-ischemia failed to protect against CI. Overall, these findings support the hypothesis of an involvement of ROS as a significant cause of ischemia-RI-induced cerebral injury, and suggest that PBN may be a useful agent for the prevention of CI—the protective effect in ischemia/RI being related to its ability to prevent a cascade of FR generation by forming spin-adducts.
In 1993, Sen and Phillis [
67] communicated that a combination of systemic and topical PBN (100 mM) was required to suppress hydroxyl radical formation during CI/RI, as PBN FR adducts were detected in EPR spectra of the lipid extracts of PBN-treated rat brain subjected to CI/RI.
In 1992, Yue and co-workers [
68] described the ability of PBN to attenuate ischemia-induced forebrain (global brain ischemia) edema and hippocampal CAl neuronal loss in gerbils and to protect rat cerebellar neurons in primary culture from glutamate-induced toxicity. Thus, PBN, given IP at 75 or 150 mg/kg 30 min before CI (5 min MCAO) increased survival (7 d) of CAl neurons from 60 ± 14 (vehicle treated, n = 17) to 95 ± 15 (
p < 0.05, n = 15) and 145 ± 3 (
p < 0.01, n = 15), respectively. When gerbils were treated with PBN (50 mg/kg, IP) immediately and 6 h after RI, followed by the same dose for an additional 2 d, CAl neurons survival improved from 35 ± 9 (vehicle, n = 20, 6 min MCAO) to 106 ± 17 (
p < 0.01, n = 13). In gerbils exposed to a more severe CI (10 min), pre-treatment with 150 mg/kg PBN increased the survival of CAl neurons from 6 ± 6 (vehicle) to 27 ± 10 (
p < 0.05, n = 11). Pretreatment with PBN, at 150 mg/kg, reduced forebrain edema (following 15 min CI) by 24.7% (
p < 0.01, n = 16). Note that PBN at 50 mg/kg, IP, had no hypothermic effect and at 75 or 150 mg/kg caused a transient hypothermia. The presence of PBN in the brain was confirmed in microdialysis samples and brain tissue extracts using HPLC. Finally, it was reported that in vitro, PBN protected rat cerebellar neurons against 100 µM glutamate-induced toxicity (EC
50 = 2.7 mM). These results further support the concept that FRs contribute to brain injury following CI and suggest the potential therapeutic application of electron spin traps in stroke.
In 1994, Zhao et al. [
69] observed that, in a transient MCAO experiment in rats, PBN dramatically reduced infarct size when given 1 or 3 h after recirculation following a 2 h period of MCAO. PBN was dissolved in saline (10 mg mL
−1 or 20 mg mL
−1) and administered intraperitoneally. Four groups of animals were studied: one injected with saline (n = 16), and three with PBN (n = 8, n = 10, and n = 11). Four doses of PBN 100 mg kg
−1 were given, with an interval of 12 h. The initial dose of PBN was either given 15 min before MCAO or 1 h or 3 h after recirculation. Eight control animals were given saline 15 min before CI and another eight animals were given saline post-ischaemically in four doses. Since the results were similar, the two control groups were pooled. Blood pressure, PO
2, PCO
2, pH, and blood glucose were measured before and after the induction of CI and at the end of the 2 h ischaemic period. Temperature was controlled during CI and for the first 4 h of recirculation. These results, and those reported by Cao and Phillis (see below) [
46], demonstrate that the effect of PBN in ameliorating infarct size following MCAO surpasses that of any other drug tested before, as no other drug reduced the infarct size to 50% of control or less when given as late as 5 h after permanent MCAO or 3 h after recirculation, following 2 h MCAO.
In 1994, Cao and Phillis [
46] reported the first study to demonstrate protection by PBN treatment against permanent focal MCAO and ipsilateral common carotid artery occlusion in rats. Studies on the metabolism and distribution of PBN in rats have shown that PBN is rapidly absorbed after IP injection in rats. The PBN level in plasma peaks at 15 min and decreases steadily over the subsequent 12 h, with a half-life in blood plasma of 2–3 h. The concentration of PBN in the brain peaks 30–45 min after administration and then decreases steadily during the next 8 h. The brain concentration of PBN is significantly higher than that in blood. The increased brain distribution of PBN was attributed to its greater lipophilicity and its ability to penetrate the blood–brain barrier (BBB). PBN was selected as the spin-trapping agent and its times of administration were scheduled based on the above results. The doses of PBN used in the present study replicated those used in previous studies by Phillis and Clough-Helfman [
65,
66]. Thus, PBN was given IP at 100 mg/kg at initial times of 0.5 h prior to CI (group 2) and 0.5 (group 3), 5 (group 4), and 12 h (group 5) after CI. Additional doses of PBN (100 mg/kg) were administered as follows: group 2 at 24 h; group 3 at 5, 17, 29, and 41 h; group 4 at 17, 29, and 41 h; group 5 at 24 and 36 h. Animals were sacrificed 48 h after MCAO and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained 1.5 mm slices of the forebrain. PBN significantly attenuated cortical infarct volume and cerebral edema in all of the treated rats compared with those in ischemic control (group 1), with no significant differences between the different PBN treated groups. The percentage of infarct volume in ischemic control rats was 22.7 ± 1.0, while those in PBN-treated groups were 9.6 ± 2.0,
p < 0.01 (group 2); 12.2 ± 2.2,
p < 0.01 (group 3); 11.1 ± 2.9,
p < 0.01 (group 4); and 14.4 ± 2.5,
p < 0.01 (group 5). Thus, these results indicate that PBN (100 mg/kg) treatment, initiated both prior to and for up to 12 h after the onset of CI, significantly reduced cortical infarct volume and brain edema at 48 h post-ischemia. Furthermore, neurological behavior tests showed that PBN decreased the neurological deficit scores (NDS) in rats initially treated either prior to or for up to 12 h after CI. There was an indication that if treatment was initiated later, following MCAO, there would be a reduction in the protective effect. Although pretreatment with PBN may appear to be therapeutically impractical, since most acute strokes occur without warning, PBN administration also had a significant neuroprotective effect when its administration was initiated 12 h after CI.
To sum up, whereas the therapeutic time window for NMDA antagonists, non-NMDA antagonists, and glutamate release inhibitors in focal models of CI appears to be about 1–2 h, PBN still showed neuroprotective efficacy even when it was administered 12 h following focal CI. These findings demonstrate that PBN has a strong neuroprotective effect, supporting the hypothesis that FRs play an important role in brain injury following CI, and suggest that PBN could have potential therapeutic value for the treatment of stroke [
46].
In 1995, Siesjö and colleagues [
70] communicated the results of a study targeted to explore whether PBN influences the recovery of energy metabolism in rats following transient focal CI. MCAO of 2 h duration was induced in rats by an intraluminal filament technique and brains were frozen in situ at the end of CI and after 1, 2, and 4 h of recirculation. PBN was given 1 h after recirculation. Neocortical focal and penumbra areas were sampled for analyses of phosphocreatine (PCr), creatine, ATP, ADP, AMP, glycogen, glucose, and lactate. The penumbra showed a moderate-to-marked decrease and the focus showed a marked decrease in PCr and ATP concentrations, a decline in the sum of adenine nucleotides, near-depletion of glycogen, and an increase in lactate concentration after 2 h of CI. Recirculation for 1 h led to only a partial recovery of energy state, with little further improvement after 2 h and signs of secondary deterioration after 4 h, particularly in the focus. After 4 h of recirculation, PBN-treated animals showed significant recovery of energy state, with ATP and lactate contents in both focus and penumbra approaching normal values.
To sum up, it was found that PBN allowed recovery of ATP and lactate contents in tissues with dense CI and low ATP values during a 2 h period. In theory, this could reflect the accumulation of PBN in mitochondria, where it could conceivably prevent FR damage to components of the respiratory chain—PBN improving the microvascular function. Thus, these results unequivocally demonstrate that PBN markedly improves the recovery of cerebral energy state and mitochondrial metabolism [
70].
In 1996, Pahlmark and Siesjö [
71] described the results of the administration of PBN in a transient forebrain CI experiment in rats, either 30 min before or 30 min or 6 h after CI, with PBN or its vehicle. Thus, after 15 min of two-vessel MCAO in anaesthetized rats, brain damage was assessed by histopathological techniques 7 d later. PBN reduced neuronal necrosis in the neo-cortex when given 30 min post-treatment but not when given before or 6 h after CI; it also failed to reduce damage to the hipocampal CA1 or the caudoputamen. This result was totally unexpected because, although PBN has little effect on neuronal damage due to forebrain CI, it has been reported that PBN dramatically reduces infarct size due to permanent or transient MCAO in the same species [
46,
69]. These results and are in disagreement with those obtained in gerbils subjected to a similar type of forebrain ischemia [
72,
73]. Thus, it is tentatively concluded that while PBN ameliorates either the microvascular dysfunction or the mitochondrial failure, which could be the crucial events leading to infarction in focal CI, it has only a weak effect on the mechanisms that yield selective neuronal necrosis in transient brief CI.
In 1997, Jenkins et al. [
74] communicated the effects of PBN on focal CI/RI in halothane-anesthetized rats after 90 min MCAO and RI. Intravenous injections of 25 mg/kg PBN 5 min before and 30 min after the insertion of a filament significantly attenuated the lesion when the volume was measured 24 h after CI. During CI and during the first 30 min after RI, cerebral blood volume and blood flow were measured by volume-sensitive and newly developed flow-sensitive magnetic resonance imaging techniques and by laser– Doppler flowmetry. In all the animals, the area of decreased blood flow was larger than the area of decreased volume by a factor of 2.2. The area of the postrefusion flow deficits matched the final lesion volumes at 24 h measured histologically much better than did the blood volume deficits on both saline and PBN-treated animals. RI led to a return of blood flow and volume to values close to the contralateral side in the PBN-treated animals, in contrast to the saline-treated control group. It was concluded that in focal CI/RI, PBN provides protection of vascular endothelium, leading to enhanced postischemic RI, a result that suggests that the vascular endothelium may be a primary target for the damaging action of FRs given the protection afforded by PBN.
In 1997, Grotta and co-workers [
75] showed that PBN reduced the damage produced by global and focal CI, with the latter being reduced even if PBN was administered 5 and 12 h after MCAO in reversible and permanent models in rats, respectively. Similar to previous reports [
46,
68,
69], Grotta et al. demonstrated that PBN administered before CI dramatically reduces RI, resulting in an infarct volume indistinguishable from that produced by permanent CI. However, in contrast to PBN pretreatment and reports on PBN neuroprotection after delayed post-treatment [
46,
69], administration of PBN 2 h after MCAO (1 h before RI) was ineffective in reducing RI in the used ischemial reperfusion model. This lack of post-treatment efficacy casts doubts on the central importance of FRs in causing RI in this model [
46] and suggests that PBN pre-treatment has a positive effect on RI by some other mechanism(s) for PBN neuroprotection. Because of the reported [
46] unusually long (12 h) window for protection with PBN using a permanent focal CI model with no RI, it is possible that, to provide ultimate neuroprotection, PBN must interact with a type of cellular processing that occurs during CI rather than during RI. For instance, it has been described that irreversible loss of Ca
2+/calmodulin-dependent protein kinase II (CaM-KIT) activity, occurring during early CI, correlated with neuronal damage after global and focal CI [
76] and that NMDA antagonists and hypothermia, which were able to decrease CaM-KII inactivation, were effective in decreasing brain damage [
77]. Interestingly, PBN has been shown to completely inhibit inactivation of CaM-KII after CI in gerbils [
78], suggesting that prevention of CaM-KIl inactivation or improvement of its recovery is one possible way that PBN may protect the brain from CI.
In 1999, Green and co-workers [
79] described their attempts to determine whether delayed treatment with PBN was protective at 2 months following transient global forebrain CI and whether additive effects can be observed when PBN is administered in combination with moderate hypothermia. For this objective, rats were subjected to 10 min of two-vessel forebrain CI followed by 3 h of postischemic normothermia (37 °C); 3 h of postischemic hypothermia (30 °C); normothermic procedures, combined with delayed injections of PBN (100 mg/kg) on days 3, 5, and 7 post-insult; and postischemic hypothermia combined with delayed PBN treatment, or sham procedures. Cognitive behavioral testing and quantitative histopathological analysis at 2 months were carried out. It was observed that postischemic PBN injections induced a systemic hypothermia (1.5–2.0 °C) that lasted for 2–2.5 h. Water maze testing revealed significant performance deficits relative to shams in the normothermic ischemic group, with the postischemic hypothermia and PBN groups showing intermediate values. A significant attenuation of cognitive deficits was observed in the animal group receiving the combination postischemic hypothermia and delayed PBN treatment. Quantitative hippocampal CA1 cell counts indicated that each of the ischemia groups exhibited significantly fewer viable CA1 neurons compared to sham controls. However, in rats receiving either delayed PBN treatment or 3 h of postischemic hypothermia, significant sparing of CA1 neurons relative to the normothermic ischemia group was observed.
To sum up, these data indicate that hypothermia combined with PBN treatment provides long-term cognitive improvement compared to non-treated groups. Thus, PBN-induced mild hypothermia could contribute to the observed neuroprotective effects [
79].
In 1999, Niwa et al. [
80] compared the effects of PBN and edaravone (
Figure 1), evaluating them in a rat transient MCAO model to clarify the possible role of ROS in the RI. The volume of cerebral infarction, induced by 2 h MCAO and subsequent 2 h RI in Fisher-344 rats, was evaluated once PBN (100 mg/kg) and edaravone (100 mg/kg), administered just before reperfusion of MCAO, had been significantly reduced by a similar percentage. Edaravone significantly prevented
•OH-induced hydroxylation of salicylate but did not influence superoxide generation; it significantly reduced the infarction volume following transient focal brain CI by a similar percentage to PBN. In these preliminary results, the same dose of edaravone significantly diminished the brain edema caused by transient CI in this model. From these observations, it was concluded that
•OH is mainly responsible for the cerebral RI after transient focal CI.
In 2000, Zausinger et al. [
49] reported that treatment with PBN significantly reduced cortical infarction (−31%) in a 90 min reversible unilateral MCAO in rats. The same team [
81] reported the observed effects in rats subjected to 90 min of MCAO in a rat model of reversible focal CI, local cerebral blood flow (LCBF) bilaterally recorded by laser Doppler flowmetry, NDSs quantified daily, and infarct volume assessed after 7 d. Thus, MCAO reduced ipsilateral LCBF to 20–30% of baseline. After RI, postischemic hyperemia was followed by a decrease in LCBF to about 70% of baseline; the improvement of neurological function and reduction of infarct volume (25%) in animals treated with PBN was not significant.
In 2000, Yang and colleagues [
82] evaluated the neuroprotective effect of PBN compared to a vehicle in a focal embolic MCAO in rats. Wistar rats were randomly divided into three groups (n = 10, each group). Animals in the control group received the vehicle and those in the treatment groups were treated with PBN, (both 100 mg/kg/d × 3 d, IP) starting 2 h after the introduction of an autologous thrombus into the right-side MCAO. The neurological outcome was observed and compared before and after treatment and between groups. The percentage of infarct volume was estimated from TTC stained coronal slices 72 h after the ischemic insult. 2 h post–ischemia administration of PBN significantly improved NDS at 24 h following MCAO embolization (both
p < 0.01). The percentage of infarct volume for animals receiving the vehicle was 32.8 ± 9.4%. 2 h-delayed administration of PBN achieved a 35.4% reduction in infarct volume in treatment groups when compared with animals receiving the vehicle (PBN vs. control, 21.2 ± 10.9% vs. 32.8 ± 9.4%;
p < 0.05) (
Figure 2). These data indicate that free radical generation may be involved in brain damage in this model and that 2 h delayed postischemia treatment with PBN may have neuroprotective effects in focal CI.
Furthermore, there was no significant difference in the attenuation of cerebral ischemic lesion between various groups with administration of PBN with an initial time of 0.5 h prior to CI and 0.5, 5, and 12 h after CI [
82].
Li and co-workers [
83], based on the potent neuroprotective effect of PBN when administered after transient focal CI, and to further elucidate the mechanism of PBN action, have studied its effect on animal survival, histopathological outcome, and activation of caspase-3 following 30 min of global CI in vehicle- and PBN-treated rats. The results showed that 30 min of global ischemia was such a severe insult that no animal could survive beyond 2 d of RI. Histopathological evaluation showed severe tissue edema and microinfarct foci in the neo-cortex and thalamus. Close to 100% damage was observed in the
stratum and hippocampal CA1 and dentate gyrus subregions. Post-ischemic PBN treatment significantly enhanced animal survival and reduced damage in the neo-cortex, thalamus, and hippocampus. Immunohistochemistry demonstrated that caspase-3 was activated following ischemia in the striatum and the neo-cortex. PBN suppressed the activation of caspase-3 in both structures. It was concluded that PBN is a potent neuroprotectant against both local and global CI. Besides its function as a FR scavenger, PBN may reduce ischemic brain damage by blocking cell death pathways that involve caspase-3 activation.
Despite the fact that PBN is a well-established and useful neuroprotective agent in CI in vivo models, to date, there is little information concerning its effectiveness when administered in combination with rtPa.
In 2001, Lapchak et al. [
84] determined the effects of PBN when administered before rtPA on hemorrhage and infarct rate and volume. A total of 165 male New Zealand white rabbits were embolized by injecting a blood clot into the MCA via a catheter. 5 min after embolization, PBN (100 mg/kg) was infused intravenously. Control rabbits received saline—the vehicle required to solubilize the spin traps. In rtPA studies, rabbits were given intravenous rtPA starting 60 min after embolization. Post-mortem analysis included the assessment of hemorrhage, infarct size and location, and clot lysis. In the control group, the hemorrhage rate after a thromboembolic stroke was 24%. The amount of hemorrhage was significantly increased to 77% if the thrombolytic rtPA was administered. In the combination drug–treated groups, the PBN/rtPA group had a 44% incidence of hemorrhage. rtPA and PBN/rtPA were similarly effective at lysing clots (49% and 44%, respectively) compared with the 5% rate of lysis in the control group. There was no significant effect of drug combinations on the rate or volume of infarcts. This study suggests that PBN may have deleterious effects when administered after an embolic stroke. However, PBN, when administered in combination with rtPA, may improve the safety of rtPA by reducing the incidence of rtPA-induced hemorrhage.
In 2003, Christensen and co-workers [
85] analyzed rats in permanent MCAO and treated 1 h after occlusion with a single dose of PBN (100 mg/kg) or saline. Body temperature was measured and controlled for the first 24 h to obtain identical temperature curves in the two groups. Cortical infarct volumes were determined on histological sections 7 d later. PBN did not significantly reduce infarct volume (control 28.3 ± 16.3 mm
3 vs. α-PBN 23.7 ± 7.4 mm
3). PBN, administered shortly following the induction of CI on infarct volume after an extended survival period of 1 week in the temperature-controlled rat model of permanent MCAO, did not reduce infarct size.
Although it has been suggested that FRs are involved in the genesis of ischemic brain damage, as shown by the protective effects of PBN in ischemic cerebral injury, Goda et al. have investigated the involvement of FRs in transient ischemic-induced delayed neuronal death [
86]. However, the protective effect of PBN on the cerebral damage caused by MCAO–reperfusion in rats is known [
69]. PBN has also been reported to reduce cortical infarct and edema in rats subjected to focal CI [
46]. When administered either 30 min prior to or 30 min post-CI, PBN significantly reduced the degree of neuronal damage and loss in the hippocampal CA1 region [
66]. These findings indicated that FRs contribute to necrotic, but not apoptotic, neuronal damage. However, Goda’s results indicate that PBN does not suppress delayed neuronal death and that FRs do not appear to contribute to hippocampal CA1 injury following transient forebrain CI [
86].