Introduction
Stroke is the second leading cause of death and the preeminent cause of neurological disability worldwide [
1‐
3]. Ischemic stroke results from the sudden decrease or loss of blood circulation to an area of the brain, resulting in a corresponding loss of neurological function. Deficits can include partial paralysis, difficulties with memory, thinking, language, and movement. While the prompt restoration of blood flow to the ischemic tissue is the current strategy of choice in clinical stroke treatment, this can cause additional damage and exacerbate neurocognitive deficits. Inflammation is a key feature of cerebral ischemia [
4,
5] with major immune system players, namely microglia [
6,
7] and mast cells [
8,
9] acting as early responders. This response leads to the production of pro-inflammatory mediators and infiltration of other inflammatory cell populations (e.g., neutrophils, T cell subsets, monocyte/macrophages) into the ischemic brain tissue. Additional, late-phase responders include reactive astrocytes and the resulting glial scar which forms in the boundary zone of the ischemic core and may play a critical role, both in detrimental and beneficial terms [
10].
Experimental animal models of stroke enabled the identification of a wide array of anti-inflammatory/neuroprotective compounds, including anti-epileptics, inhibitors of inducible nitric oxide synthase and kinases, minocycline, antioxidants, and polyphenols [
11‐
15]. Some of these putative neuroprotectants have been tested in human clinical trials, but they have yielded little positive outcome [
3,
16]. Nevertheless, neuroprotection studies utilizing animal models still provide useful insights into strategies for limiting stroke severity as they continue to offer translational potential for improving future stroke outcome [
3,
17]. The failure of therapies targeted only to neuronal cell protection may be attributable, in part, to a lack of concomitant protection of cerebral blood vessels from secondary injury by inflammation and reactive oxygen species/reactive nitrogen species. Although anti-inflammatory approaches have proven successful in animal models of stroke [
18,
19], attempts to translate this into clinical application have fallen short of expectations [
20,
21].
In response to tissue injury and stress, the body is known to respond by producing molecules “on demand” which function to restore homeostatic balance and prevent further damage [
22]. Among these is a class of lipid signaling molecules, the
N-acylethanolamines (NAEs) [
23,
24]. One NAE, in particular
N-palmitoylethanolamine (PEA or palmitoylethanolamide), is surrounded by a large number of observations supporting its role in maintaining cellular homeostasis by acting as a mediator of resolution of inflammatory processes [
25,
26]. These past years have witnessed a continually growing number of studies confirming the anti-neuroinflammatory and neuroprotective actions of PEA [
27‐
30]. Interestingly, several recent studies have shown that a co-ultramicronized PEA/luteolin composite (co-ultraPEALut, 10:1 mass ratio) is more efficacious than PEA alone [
31,
32], including animal models of spinal cord injury [
33] and traumatic brain injury [
34].
Based on the above observations, we carried out a two-part study with co-ultraPEALut in cerebral ischemia. In the first part of the study, we analyzed the neuroprotective and anti-inflammatory properties of co-ultraPEALut in a rat model of middle cerebral artery occlusion (MCAo), while in the second part, the effects of co-ultraPEALut (Glialia®) were assessed in stroke patients undergoing rehabilitation therapy. We chose to conduct the study in subacute-phase stroke patients, given that even at this stage, certain characteristic pathological features, in particular neuro-inflammatory processes, are still active and able to cause continued neuronal cell damage. Moreover, many patients with ischemic stroke, despite optimal medical treatment received during the acute phase, often fail to recover (or only partially), leading to persistent disability requiring rehabilitation. As Glialia® is already a marketed product, we investigated whether treatment with Glialia®, carried out simultaneously with rehabilitation therapy, can bring about a better functional recovery in stroke patients in the subacute phase over a prolonged time frame.
Discussion
Cerebral ischemia continues to represent one of the principal unmet medical needs in today’s society. Stroke is especially devastating, given that it constitutes the most frequent cause of neurological disability worldwide. The underlying cellular mechanisms of stroke neuropathology are complex. An initial episode of focal hypoperfusion subsequently leads to excitotoxicity, oxidative damage, microvascular injury, blood–brain barrier dysfunction, and post-ischemic inflammation [
4,
49‐
51]. Despite almost four decades of experimental animal investigations on stroke and the identification of a spectrum of anti-inflammatory/neuroprotective compounds [
11‐
15], translatability of these findings to human clinical trials until now have been proven uniformly disappointing [
3,
16]. In the present study, we describe the neuroprotective effects of co-ultramicronized palmitoylethanolamide/luteolin in a rat model of focal cerebral ischemia and, more importantly, the ability of co-ultraPEALut (Glialia®) to improve the neurological status of stroke patients undergoing neurorehabilitation.
Focal cerebral ischemia is accompanied by reactive astrogliosis [
52] and activation of microglial cells in the hippocampal area [
53]. Reactive gliosis can lead to the production of excessive amounts of cytokines as well as inflammatory products that exacerbate ischemic damage [
54]. In our study, an increased in GFAP immunoreactivity was observed in the MCAo group in comparison to sham-operated animals while there was a marked reduction in the co-ultraPEALut-treated group. As previously reported [
55], co-ultraPEALut is able to significantly reduce expression levels of GFAP after MCAo. In the current MCAo model, oral administration of co-ultraPEALut 1 h after ischemia and 6 h after reperfusion improved neurological score, reduced lesion size and histological damage, inhibited mast cell infiltration/degranulation and astrocyte activation (as measured by GFAP accumulation, a characteristic neuropathologic feature of ischemic brain injury [
56]), and restored expression of BDNF and GDNF. Experimental and clinical studies have shown that BDNF and GDNF are upregulated at very early stages during brain ischemia [
56]. Furthermore, exogenous administration of GDNF and BDNF reduced the toxic effects of excitatory amino acids, attenuated nitric oxide production, and lowered apoptosis/cell death in stroke animal models [
56,
57]. In our preclinical findings, protein analysis showed that both BDNF and GFAP levels were downregulated by cerebral ischemia while a local and sustained increase in their expression in the perilesioned tissue followed oral administration of co-ultraPEALut.
Mast cell activation and degranulation is known to contribute to blood–brain barrier disruption in cerebral ischemia [
58]. Other elements play a role in ischemic brain damage, such as activation of the transcription factor nuclear factor-κB [
59,
60], inducible nitric oxide synthase [
61,
62], and poly(ADP-ribose)synthetase [
63]; increased expression of the pro-apoptotic protein Bax [
64]; and decreased expression of the anti-apoptotic molecule Bcl-2 [
65]. In the present study, co-ultraPEALut significantly reduced nuclear factor-κB translocation, attenuated poly(ADP-ribose)synthetase activation, and normalized Bax/Bcl-2 expression levels. These results are qualitatively similar to our earlier investigations with PEA alone, although in the latter, 10-fold higher doses of PEA (compared to co-ultraPEALut) were required for efficacy [
55,
66].
PEA actions are mediated, at least in part, by activation of peroxisome proliferator-activated receptors, accompanied by a decrease in neutrophil influx and expression of pro-inflammatory proteins, such as inducible nitric oxide synthase and cyclooxygenase-2 [
39,
67]. Luteolin displays specific anti-inflammatory effects, which are only partly explained by its antioxidant capacities. The anti-inflammatory activity of luteolin includes activation of antioxidative enzymes, suppression of the nuclear factor-κB pathway, and inhibition of pro-inflammatory substances [
68,
69]. The molecular basis behind the superior pharmacological efficacy of co-ultraPEALut compared to comparable concentrations of the single chemical components is currently under investigation.
Although inflammatory signaling is considered to participate in the early post-ischemic period, as the ischemic cascade progresses, cell death leads to a new phase of the inflammatory response, whereby the immune system becomes activated. There is evidence that both microglia and mast cells have a role at later times following the ischemic episode. Mast cells possess a potent armamentarium to target the components of the blood–brain barrier and basal lamina shortly after their activation in ischemia, whereas de novo production of mediators reactivates and maintains the process over the longer term [
70‐
73]. Further, a newly published study demonstrates that mast cells can undergo a delayed and long-term activation following traumatic brain injury [
74]. While the latter condition is not stroke, this report demonstrates that mast cells are not only early responders but also long-term players in brain injury. Further, it bears keeping in mind that in terms of
acute versus
late-stage treatment regimens in rats and man, one cannot necessarily make a direct comparison between time scales. In other words, a patient treated starting 60 days post-stroke does not have a rat equivalent.
Despite literally hundreds of compounds and interventions that provide benefit in experimental models of cerebral ischemia, efficacy in humans remains to be demonstrated [
75]. Many patients with ischemic stroke, despite optimal medical treatment received during the acute phase, often fail to recover (or only partially), leading to persistent disability requiring rehabilitation. As Glialia® is already a marketed product, we investigated whether treatment with Glialia®, carried out simultaneously with rehabilitation therapy, can bring about a better functional recovery in stroke patients in the subacute phase. The observations reported here demonstrate that a positive outcome in an animal stroke model can be translated into stroke patients. Open studies, however, necessarily impose certain limitations as, for example, the lack of a randomized controlled trial’s robustness and the absence of a control group. Further, outcome of an observational study may be biased by patient attributes that affect treatment selection. Formal clinical trials typically employ randomization to address some of these issues by balancing baseline characteristics among the treatment groups. This open study was intended to consider patients who are observed in the context of neurological disability resulting from cerebral ischemia and undergoing rehabilitation—independent of their gravity. The large patient base provided a range in times from initial ischemic episode until the beginning of treatment with Glialia® and can serve to gauge the evolution of a patient’s disability with treatment in a long-term rehabilitation setting with the same rehabilitation team. We believe that such information may allow one to derive considerations for clinical practice on choice of therapy to combine with rehabilitation for patients with persistent stroke-related neurological disabilities.
Systematic reviews comparing the results of randomized controlled trials and observational studies of the same agents have failed to demonstrate significant differences in outcomes across multiple study designs [
76]. In order to address the absence of a control patient group, we compared the results obtained by the CNS, Ashworth Scale, and Barthel Index in patients treated with Glialia® with historical literature [
77] data observed in patients having similar pathologic conditions but never receiving Glialia® (Table
5). A
z test comparing the mean value reported (standard error was not given) and that of the present study was performed; this comparison is limited to the observed mean value and a fixed value. As such, the test tends to overestimate the probability value and therefore was fixed (alpha = 0.01).
Table 5
Comparison of current multicenter study results with reported values for stroke patients not receiving Glialia®
Canadian Neurological Scale | 1.80 | 0.01 (p < 0.0001) | 1.02 (p < 0.0001) |
Ashworth Scale | −0.20 | – | 0.58 (p < 0.0009) |
Barthel Index | 36.2 | 0.62 (p < 0.0001) | 34.8 n.s. |
These caveats aside, this represents the first description of co-ultraPEALut administration to human stroke patients and clinical improvement not otherwise expected from spontaneous recovery. Based on these observations, we believe that controlled trials are warranted to confirm the utility of co-ultraPEALut to improve clinical outcome in human stroke, also in consideration of the excellent tolerability associated with Glialia® treatment. A double-blind, randomized, and placebo-controlled trial of Glialia® in stroke patients within 12 h of the initial ischemic episode has been initiated.