**3. Copolymer-1**

*Neuroprotection - New Approaches and Prospects*

ecules that induce necrosis and apoptosis [9].

**2. Immunological response in stroke**

the integrity of the BBB.

IL-10, and arginase 1 [23].

stroke [18].

necrosis factor-α (TNF-α) by endothelial cells [10].

lium seeking to protect of the blood brain barrier (BBB) [12].

depolarization of the membranes causing the intracellular increase of Ca2+ that is added to the one released by the endoplasmic reticulum and mitochondria [6].

Neuronal depolarization causes the release of glutamate which, when bound to its ionotropic N-methyl-D-aspartate (NMDA) and -amino-3-hydroxy-5-methyl-4-isoxazolpropionic (AMPA) receptors, achieves greater depolarization and, as a consequence, conditions of excitotoxicity [7]. These conditions are coupled with the production of free radicals [8] and lead to cell death by the activation of mol-

Along with the lesion caused by the decrease in blood flow, the immune response is added to the events involved in both the detriment of the tissue and its protection.

Inflammation is usually present before the development of arterial obstruction that gives rise to the ischemic event. The development of atherosclerosis is accompanied by the production of oxygen free radicals (ROS), expression of cell adhesion molecules, and production of proinflammatory cytokines as IL-1β and tumor

Shortly after occlusion, endothelial cells express a greater amount of intercellular adhesion molecules (ICAM), deposition of mannose binding lectin molecules that trigger activation of the complement pathway [11], producing higher amounts of ROS. The overproduction of ROS activates the prostaglandin pathway that stimulates the production of matrix metalloproteinases (MMP) that even though degrading constituents of the extracellular matrix, reshape the vascular endothe-

The release of chemokines such as CCL2 allows endothelial permeability [13], leading to the translocation of P-selectin from Weibel-Palade bodies, as well as the expression of ICAM-1 and vascular cell adhesion molecule (VCAM)-1 and E-selectin, on the endothelial surface [14]. Theses phenomena, together with the damage of the extracellular matrix facilitate the extravasation of macromolecules and water, which causes the development of vasogenic edema [15]. Peripheral immune cells then enter the injured cerebral parenchyma [16] facilitating the loss of

Neutrophils are the first leukocytes that migrate to the cerebral parenchyma; they have been detected since the first hour after ischemia and reach their maximum peak in 1–3 days [17]. In the clinic, it has been observed that the higher blood neutrophil count is associated with higher infarction volumes in patients with acute

The second cell type that enters the neural tissue are monocytes, these infiltrate within 24 h of the onset of the ischemic event reaching its peak on day 3 [19]; their differentiation process toward macrophages and their activation will be determined by the molecular environment to which they arrive. This process is similar to that experienced by T lymphocytes, which reach the parenchyma 24–96 h post-ischemia [20]. At the same time, the cells of the injured cerebral parenchyma release damage associated molecular patterns (DAMPs) that activate the microglia. Depending on the activation environment, the microglia can acquire a proinflammatory (M1) or anti-inflammatory (M2) phenotype [21]. In the M1 phenotype, the microglia acquires phagocytic capacity, produces NO, free radicals, and proinflammatory cytokines (e.g. TNF-α, IL-12 and IL-6) [22]. Some regions in the ischemic penumbra present an activation of M2 microglia distinguished by the production of anti-inflammatory and repair molecules, such as insulin growth factor 1 (IGF-1),

**144**

Copolymer-1 [Cop-1], also known as glatiramer acetate (GA) or copaxone [trade name], is a blend of peptides formed by random sequences of four amino acids: glutamic acid, lysine, alanine, and tyrosine; these have a variable length from 45 to 200 amino acid residues and a molecular weight of 4000–9000 Da [33].

Cop-1 was originally synthesized from mylelin basic protein (MBP) to identify the precise immunogenic sequence and provoke experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS); however, it did not present encephalitogenic characteristics [34]; on the contrary, it has suppressive and protective effects on EAE [35]. In the clinic, copaxone is able to diminish the relapse rate and improve the disability of patients with relapsing-remitting MS [36]. Copaxone obtained its approvement by the Food and Drugs Administration [FDA] of U.S.A. in 1996 and in Europe in 2001 [33].

At this time, the exact mechanism by which Cop-1 exerts its protective effects is not known at all. Studies carried out in EAE suggest that Cop-1 has greater affinity for the MHCII binding site of APC when competing with peptide complexes derived from the MBP, specifically with the epitope 82–100 [37]. This competition may also be present among the complexes for the TCR binding site of the lymphocytes [38] that, when activated, induces a Th2 response [39].

The Cop-1 response is distinguished by increased synthesis of IL-4, IL-5, IL-10, IL-13, and TGF-β [33, 40–43]. Cop-1 has also been observed to increase the presence of regulatory T lymphocytes [44] and regulatory CD8+ T lymphocytes in patients with multiple sclerosis [45, 46].

Another important effect of copolymer-1 is the production of growth factors, among which stand out; the brain derived neurotrophic factor [BDNF] [47, 48], IGF-1, [49] and neurotrophins NT-3 and NT-4 [47]. It is known that, in addition to inducing neuroprotection and neurorestoration, these growth factors are related to mechanisms such as memory and learning.

The molecular basis by which Cop-1 exerts its neuroprotective effect has been evaluated in several *in-vitro* assays. The most explanatory results have been obtained in the analysis of the effect of Cop-1 on APC such as monocytes, microglia, and astrocytes.

It has been showed that through the blockade of the nuclear factor kappa B [NFkB], Cop-1 reduces the expression of the chemokine CCL5 [RANTES], which is upregulated by the presence of IL-1β [50] and TNF-α in human astroglial cells [51]. A similar effect has also been observed on the monocyte chemotactic protein-1 [MPC-1] and adhesion molecules VCAM-1 and selectin E in endothelial cells as well as COX2 and iNOS [52].

It has also been observed that Cop-1 induces differentiation of type II monocytes independently of the binding of Cop-1 to MHCII. Weber et al. demonstrated that this differentiation is due to the fact that Cop-1 reduces the phosphorylation of the transcription factor STAT-1 by stimulating the expression of IL-10 and TGF-β [53].

On the other hand, it has also been observed that Cop-1 has a direct effect on glial cells [microglia and astrocytes] which are activated in conjunction with T cells reducing STAT-1 and STAT-3 phosphorylation through increased expression of cytokine signaling suppressor (SOCS-1) and independently of IFNϒR, accompanied by a reduction of IL-12 by CD4+ T lymphocytes [54].

Even though the molecular pathways by which Cop-1 acts are not yet completely established, the microenvironment induced by this compound is capable of allowing neuroprotection since it reduces the deleterious scenario that leads to neural death. Additionally, the new conditions could facilitate tissue restoration through the synthesis of growth factors.
