Current Synthesis and Systematic Review of Main Effects of Calf Blood Deproteinized Medicine (Actovegin®) in Ischemic Stroke

Background: Stroke is one of the largest problems and clinical-social challenges within neurology and, in general, pathology. Here, we briefly reviewed the main pathophysiological mechanisms of ischemic stroke, which represent targets for medical interventions, including for a calf blood deproteinized hemodialysate/ultrafiltrate. Methods: We conducted a systematic review of current related literature concerning the effects of Actovegin®, of mainly the pleiotropic type, applied to the injury pathways of ischemic stroke. Results: The bibliographic resources regarding the use of Actovegin® in ischemic stroke are scarce. The main Actovegin® actions refer to the ischemic stroke lesion items’ ensemble, targeting tissue oxidation, energy metabolism, and glucose availability through their augmentation, combating ischemic processes and oxidative stress, and decreasing inflammation (including with modulatory connotations, by the nuclear factor-κB pathway) and apoptosis-like processes, counteracting them by mitigating the caspase-3 activation induced by amyloid β-peptides. Conclusion: Since no available therapeutic agents are capable of curing the central nervous system’s lesions, any contribution, such as that of Actovegin® (with consideration of a positive balance between benefits and risks), is worthy of further study and periodic reappraisal, including investigation into further connected aspects.


Background
The term stroke is defined as " . . . a neurological deficit attributed to an acute focal injury of the central nervous system (CNS) by a vascular cause, including cerebral infarction, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) . . . " [1], thus comprising an intraluminal obstructive/ischemic and/or wall tear and a lesion mechanism [2]. Generally, although with nuances mostly depending on the period when a specific study was undertaken, like vasculitis [20,23,24]. The atherosclerotic lesions of the vascular walls are also considered to be of inflammatory origin: leukocyte local infiltration, proinflammatory cytokines, and adhesion molecule release, which favor monocyte and T-lymphocyte endothelial adherence and lead to subsequent penetration and maintenance of a continuous inflammatory status [23,25] and/or a systemically infectious origin (Chlamydia pneumoniae, Cytomegalovirus, and Helicobacter pylori) [25]. Consequently, after blood supply arrest, a succession of extremely complex and intertwined pathophysiological processes begins within seconds [10], both detrimental and as part of the recovery, which may continue for weeks, months, or years, until reaching a clinical-evolutive relative plateau [26]. These processes are emphasized briefly below.
Abrupt and relatively prolonged deprivation of blood flow, i.e., oxygen and energetic (basically, glucose) support, leads to production collapse and drastic shortage, especially of the metabolically produced principal molecular storage and energy provider, ATP, in the most-affected brain tissue. Such severe biochemical injury generates an important amount of direct necrotic cell deaths in the core of the ischemic zone, in part because the membranes' functional and structural integrity can no longer be sustained. Being energy-dependent, resting and excitation neuronal states, which are both membrane-active processes, are markedly altered, resulting in local still-living cells dying or being at increased risk of dying (although this may be remedied if irrigation is restored sufficiently quickly). This collateral perfusion occurs in the ischemic penumbra of the infarct's periphery: (1) in neural control disturbance/abolishment of various types and severities and over different directly and/or indirectly connected territories and (2) in enhanced, inappropriate, and detrimental inner bio-pathologic augmented activity with enhanced ATP consumption, which is already diminished [10,19,27].
If the blood flow arrest continues without sufficient collateral flux supply and within cerebrovascular autoregulation [22] or in the peripherally situated ischemic penumbra, further injuries may occur, including, at the intimate level, disturbance of mitochondrial functionality with a consequently imbalanced ratio between pro-and antioxidant factors (including related scavengers) in favor of the former). Oxidative (or nitrosative) stress is mainly generated by the highly enhanced production of reactive oxygen species (ROS), generally associated with depletion but with time-dependent sequential nuances, instead providing a gene-coded transcription-factor-mediated activation of an endogenous-related defense capability. The antioxidant-response elements (AREs) of antioxidants, such as l-c-γ-glutamyl-l-cysteinylglycine (glutathione (GSH)), are highly important [28][29][30][31][32][33][34]. Subsequently, lipid peroxidation, together with phospholipases, also affect the membranes' integrity. Other critical damage actions of ROS include augmentation of the Ca 2+ intracellular amount, cytoskeleton, and DNA insults with protein oxidation [35,36], proclivity to secondary misfolding [19], enhanced involvement by gene expression activation of nuclear factor-κβ (NF-κB) of proinflammatory cytokines (chemokines and interleukins) and adhesion molecules (expressed by activated endothelial cells, which attract and stimulate the tissue plasminogen activator (t-PA) and are also considered to have therapeutic capabilities, as recombinant tissue plasminogen activator; rtPA) [37,38], and the stimulation of matrix metalloproteinases (MMPs) and other (metallo) proteases [22,39].
First, soon after ischemia is installed, neutrophils infiltrate (chemotactism) and injure the blood-brain barrier (BBB). A modern, expanded, more complex, and related conceptual structuring is the neurovascular unit that physiologically entails, by cell-cell signaling and interactions, the coordinated and efficacious comprehension and functioning of the BBB location, neurons, microglia, astrocytes, pericytes, endothelial smooth muscle cells, and intrinsic matrix proteins, and which has the adaptive capability to dynamically modify itself according to and within morphological-functional changes during post-stroke partial recovery [40,41]. Subsequently, macrophages and lymphocytes, including T cytotoxic (natural killer; NK) and B types [36], enter the damaged cerebral tissue within the above-mentioned inflammation context. In addition to those already noted, the related primum movens dwells in the signals represented by the modified osmolarity [42] and consistency of the slack post-occlusion blood, addressed to the local endothelial structure and thrombocytes [37]. Additionally involved in different but interlinked pathophysiological-related sequences are leukotrienes; growth factors; prostaglandins; astrocytes; further cell adhesion molecules, e.g., selectins; intercellular adhesion molecule 1 (ICAM-1); vascular cell adhesion molecule 1 (VCAM-1); and integrins [10,17,22,36], including with and through microglial cells that are resident in the CNS and partially transformed into phagocytes [37]. Different inflammatory pathways, some of which are respiratory [19], are also stimulated by accumulation of necrotic debris in the focal ischemic zone [39,43].
Consequent to hypoxia, the complex pathophysiological context of the ischemic stroke partially and briefly outlined above also entails acidosis, which is metabolically induced in local hypo-or anoxic circumstances, with the accumulation of lactate and hydrogen ions (H + ); the latter stimulates the production of ferrous iron-mediated ROS [10,27,36,38]. The major pathways for cell deaths are apoptosis (type I) and apoptosis-like/anoikis, autophagy (type II), and necrosis (type III) [19,41,44]. "Brain infarction was traditionally considered to be a classic example of liquefactive necrosis" [45] that can supervene quickly and brutally within a few minutes after severe and prolonged brain ischemia in the cerebral tissue, which has low tolerance to hypoxemia, such that necrosis is prone to be augmented by further pathophysiological mechanisms [46] via osmolar overload and consequent osmolysis, especially if suddenly installed [19,42]. However, a similar irreversible outcome, i.e., cell death, may also result following the other linked pathophysiological secondary injury events (summarized above) but more slowly. These latter delayed deadly damages nonetheless offer a time window for the at-risk biological structures to be rescued, at least partially [10], by spontaneous processes (prompt reperfusion, mainly based on efficient collateral blood supply restoration and vessel repermeabilization) and/or interventions. Within a major ischemic stroke, except for the overall successfully achieved (rtPA) thrombolysis, such favorable inner natural evolutions or outcomes usually do not prevail. Thereby, apoptosis and apoptosis-like phenomena also occur, including concomitantly. The former, apoptosis, is the classic pattern of programmed cell death. It often entails the mediated destruction of caspases via its propensity for phagocytosis cells to break up in connection with nuclear condensation. This may run on the intrinsic [10,29,45] mitochondrial pathway based on the release signaling of cytochrome c (a key component in the respiratory chain) and endonuclease G by proteins such as Bad, Bak, Bax, Bid, and Bim and/or those involved in metabolic pathway regulation and membrane lipids. Apoptosis may also involve permeability transition pore openings in the inner membrane components that mainly contribute to the mitochondrial outer membranes permeabilization (MOMP) [36,45,47,48]. Apoptosis targets connected enzymes such as poly-ADP-ribose-polymerase (PARP), which is, with important sex differences in its effects and with consequent nuclear DNA damage and/or exit from the mitochondria, entrance into the intracellular fluid, and continued translocation into the nucleus of the apoptosis-inducing factor (AIF), considered as being caspase-independent [36,49].
An additional pathway, named anoikis, involves the detachment of cells from the extracellular matrix (ECM) [53], including mainly with " . . . MMP-induced proteolysis of the neurovascular matrix . . . " [41]. This may also lead to programed cell death soon after stroke onset, consequent to the BBB deterioration. Specifically, this occurs due to the neurovascular unit's morphological impairment, and its secondary disfunction regards the signaling of the related inter-cells with their ECM [19,41].
Notably, in brain ischemia, necrosis, and different types of apoptosis/programed cell death, the same neuron may be affected simultaneously by caspases, calpains, and cathepsins [45].
Considering the vast complexity of CNS lesions, including in post-ischemic stroke, a justified goal for their treatment is drugs able to efficiently protect against the above-described disastrous injury developments and to provide compensatory and even recovery stimulation capabilities, as detailed below.

Methods
Given the justification noted above, an available pharmacological neuro-/bio-trophic compound with pleiotropic action, applicable to the treatment of ischemic stroke, is the calf blood deproteinized ultrafiltrate/hemodialysate, Actovegin ® (Takeda Austria GmbH, Linz, Austria).
Our goal was to target and elucidate Actovegin ® 's well-known beneficial and therapeutic effects that either directly or indirectly interfere with the morbidity pathways of ischemic stroke. These were expressly selected and synthesized in Section 1 within the complex and complicated intermingling between the damage mechanisms (DMs) [19,46] and endogenous defense activity (EDA) [54] (see Section 4).
Accordingly, we conducted a systematic literature review regarding the use of Actovegin ® in stroke. For this purpose, we searched for and interrogated related articles for the period 1 January, 2001-31 December, 2019 in reputable international medical databases: National Center for Biotechnology Information (NCBI)/PubMed, NCBI/PubMed Central (PMC) [55], Elsevier [56], Physiotherapy Evidence Database (PEDro) [57], and Institute for Scientific Information (ISI) Web of Knowledge/Science [58] (via ISI Thomson Reuters index check). In searching, we used specific sets of keywords: ("stroke", "Actovegin"/"stroke", and "calf blood deproteinized hemodialysate"/"stroke", "calf blood deproteinized ultrafiltrate"/"stroke", "Actovegin", "pleiotropic"/"stroke", "calf blood deproteinized hemodialysate", "pleiotropic"/"stroke", "calf blood deproteinized ultrafiltrate", and "pleiotropic"). Table 1 shows the numerical results of our search, which was based on a focused, step-by-step classification according to the stages of the largely used literature identification and selection method, Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (see Figure 1) [59]. Within the PRISMA paradigm, we considered only open-access/free full-text articles written in English and indexed in the ISI Thomson Reuters database. To quantify the scientific impact/indirect quality classification of each of the articles remaining after ISI checking and duplicates were removed, a custom dedicated evaluation algorithm was used [60]: first, the year of publication was considered together with the total number of citations to compute the average number of citations per year;  "stroke", "Actovegin" 0 0 16 0 "stroke", "calf blood deproteinized ultrafiltrate" 0 0 0 0 "stroke", "calf blood deproteinized", "hemodialysate" 0 0 0 0 "stroke", "Actovegin", "pleiotropic" 0 0 7 0 "stroke", "calf blood deproteinized ultrafiltrate", "pleiotropic" 0 0 0 0 "stroke", "calf blood deproteinized", "hemodialysate", "pleiotropic" Within the PRISMA paradigm, we considered only open-access/free full-text articles written in English and indexed in the ISI Thomson Reuters database. To quantify the scientific impact/indirect quality classification of each of the articles remaining after ISI checking and duplicates were removed, a custom dedicated evaluation algorithm was used [60]: first, the year of publication was considered together with the total number of citations to compute the average number of citations per year; secondly, the final PEDro score [57] for each article considered was calculated via a weighted average formula; lastly, we retained only those articles that obtained a score of at least 4 (see Table 2 for details). Thus, we eventually found five articles whose content was closest to our query, revealing that works regarding Actovegin ® and its use in stroke treatment are rather scarce; therefore, the subject matter we have chosen is worthy of investigation.
Notably, we read all of the articles identified by keywords, even if they did not qualify according to our customized PEDro-inspired indirect quality classification (see Table A1). In addition, we purchased articles that were not open-access/free full-text but that contained valuable related information to include in the study.
Despite the systematic review rigorous selection filter we have applied, according to the above-mentioned criteria-based classification methodology, some of the works related to this subject still might be missed [60]. Thus, with the aim for the search to be as exhaustive as possible, we used a mixed course of collecting the necessary bibliographic resources via standardized (the systematic literature review) and nonstandardized methods (i.e., considering related papers, including in the Romanian language) (see Table A2).
Studies undertaken since the 1960s [71] on Actovegin ® emphasized its biotrophic actions, including on skin and subcutaneous tissue lesions and/or those consequent to favorably influencing sanguine flow [66]. More recent literature, mainly since the early 2000s, also highlighted the beneficial effects of this drug on the different pathological pathways of neurological and/or vascular diseases. It "was recently demonstrated to have neuroprotective effects on neurons by increasing neuron and synaptic numbers . . . " [66]. In vitro studies indicated possible neuroregenerative effects by increasing the neurite length and the number of neuronal and excitatory synaptic contacts [62,67,69,72].
Based on studies of animal models, the actions of Actovegin ® were documented at the intimate level, including its interference with some important pathways of ischemic stroke pathophysiology and other conditions of the nervous system, either traumatic or degenerative, and central and peripheral, including diabetic polyneuropathy and endothelial dysfunction [62,65,66,72].
In more detail, Actovegin ® improves tissue oxygen and glucose consumption and energy production, for instance, in the hippocampus area, which is linked mainly to functions such as spatial learning and memory [73]. The main consequence is the increase in the related cellular metabolism by enhancing mitochondrial capacity, which has a positive effect on glucose carrier activity and oxidation, related to pyruvate dehydrogenase [62,66,67,70,72]. Thus, Actovegin ® also has insulin-like activity (ILA) [72]; IPOs may positively regulate glucose use through the activity of BBB transporters (i.e., the uniporter proteins glucose transporter 1 (GLUT1) and glucose transporter 4 (GLUT4) [66,70].
Oxidative stress is decreased by Actovegin ® [61,62,65,66,69,73] through inhibiting the nuclear enzyme poly ADP ribose polymerase (PARP) [66,68], which can detect and repair DNA damage under normal conditions but can simultaneously compromise glycolysis and mitochondria respiration, leading eventually to cellular death [66]. Using in vitro studies of rat primary hippocampal neurons, Elmlinger et al. [62] reported beneficial effects of this drug via diminishing their inner level of ROS. This dose-dependent effect was also found in in vivo studies after treatment with Actovegin ® [62,66,67,73].
Actovegin ® is involved in inflammation pathways though the activation/modulation of NF-κB with, in addition to the above-mentioned detrimental actions, a beneficial role in neuroprotection mediated by proinflammatory cytokines such as TNF-α [65,66,69,73].
In the last decade, this calf blood deproteinized hemodialysate's beneficial effects were studied in trials targeting chronic conditions such as diabetic polyneuropathy [72] and post-stroke cognitive impairment (PSCI) [63]. Notably, "stroke survivors are at increased risk of developing cognitive impairment" [61].
The concept of disease modifiers in vascular cognitive impairment (VCI) was highlighted within the 9th International Congress on Vascular Dementia in Ljubljana, Slovenia, 2015 [64]. According to one of its conclusions, Actovegin ® can be included in the above-mentioned category of pharmacological agents, including as an adequate medicine for the treatment of this mental disturbance [61,64,67].
Type 2 diabetes, diabetic polyneuropathy, and stroke should be mentioned due to their clinical importance and their frequent coexistence and related complex therapeutic approaches. Diabetes mellites (DM) is associated with an increased risk of mild cognitive impairment, dementia, and stroke [65]. Briefly, although Actovegin ® 's beneficial action pathways in diabetic polyneuropathy are not yet sufficiently understood, in addition to the factors noted above, the literature refers to its capability to "improve the cellular energy level, enhance glucose uptake and metabolism" [72]; foster "stimulation of glucose transport, pyruvate dehydrogenase, and glucose oxidation . . . " [62]; and "increase oxygen absorption and utilisation" [72].
Considering that epilepsy is a possible, mainly delayed, complication after stroke [74], Actovegin ® is a neuro-/bio-trophic for which such a condition is not among the contraindications [75].

Discussion
A modern paradigm for medication related to the above analysis asserts that " . . . using neuroprotective molecule with only one mechanism of action in disease treatment is a utopist idea . . . " [46]. Therefore, it is currently considered to be possible to provide better outcomes using therapeutic agents with pleiotropic or multimodal actions, although it is difficult to establish an immutable border in multipotential medicines between such properties [17], especially given the dynamic understanding of their effects due to a growing knowledge about this complicated domain. Specifically, " . . . a neuroprotective pleiotropic effect is related to DM (o.n.: i.e., the bundle of lesion events, as summarized above, ranging from excitotoxicity-and more-to individual genetic particularities of the post-insult response [19,46]) and represents the capacity of a pharmacological agent to interfere in more than one pathophysiological process" [54], whereas a multimodal effect refers to a drug's " . . . capacity to simultaneously regulate, in the post-lesional brain, two or more endogenous neurobiological processes of EDA . . . " [54] (i.e., mainly encompassing four basic neurobiological continuous functions within the nervous system-neuroprotection, neurotrophicity, neuroplasticity, and neurogenesis-in both physiological (especially for the latter three) and pathological circumstances).
The subtle and insufficiently deciphered interferences and partial overlaps between EDA and the not exclusively detrimental (see below) consequences of DM should be specified. This includes some EDA components; for instance, excess neuroplasticity may result in neuropathic pain, movement disorders, etc. [46]. Another example is inflammation that seems to act ambivalently and participate in " . . . oligodendrocyte cell death and contribute to demyelination after stroke or TBI . . . " [76]. However, it also has a regenerative connotation, stimulating remyelination, and neurogenesis, angiogenesis, synaptogenesis, and axonal sprouting [77].
Concerning the sophisticated conditionings between EDA components, we briefly note some more recently emphasized connections and differences (perhaps competing) within the time frame of the progress of post-insult brain tissue reactions. Neuroprotection prevails in the first hours or days, opposing "cell death, inflammation, and scarring" ( [26] and [78]; the latter is followed by "tissue reorganization" [78]. It is also partially overlapped at its inception after an elapsed period of tens of hours to a few days, with neuroplasticity and the functions of neurotrophicity and neurogenesis, as much as possible in this difficult and biologically challenging and harsh context. These are all intermingled between and with DM evolutions [46,54] toward "improving impairment/disability" [78] and "function" [26]. This extends over weeks, months, and, potentially, years [26,78], essentially referring to rehabilitation and neural repair in the subacute phase and to goal-specific training and repair in the chronic phase [78].

Conclusions
At present, no treatment intervention is available to definitively heal CNS lesions [79,80]. However, affected patients need the best possible clinical management, even under the range of weak current therapeutic options. Such patients include those affected by the frequent and disabling pathology domain represented by stroke. Therefore, further study of the literature is warranted of the possible use of any therapeutic agents that have produced favorable, albeit limited, results, including those providing small functional gains [81]. Amongst the treatments deserving further study and periodic reassessment is the Actovegin ® compound, which is a pharmacological medicine with " . . . pleiotropic, neuroprotective, and metabolic effects . . . " that " . . . fits this future vision of an integrated treatment paradigm" [66], related possible multimodal therapeutic effects being also worth being checked going forward.