**5. Effect of copolymer-1 on stroke**

As copolymer-1 has been shown to have beneficial effects in various models where neuroinflammation is a detrimental determinant, our group decided to evaluate its neuroprotective effect on cerebral ischemia. For this purpose, we used the median transient cerebral artery obstruction (tMCAO) model. Sprague-Dawley male rats were used. After being subjected to ischemia for 90 min, the rats were immunized in the interscapular region with a dose of 200 μg of Cop-1.

In the first study, the animals were evaluated for neurological deficit at day 1 and day 7 post-ischemia using the Zea Longa scale [74]. Then, a histological analysis was performed using hematoxylin and eosin staining to determine neuroprotection. The results indicated that Cop-1 is able to avoid up to 85.1% increase in infarct size (4.8 ± 1.5 for Cop-1 vs. 32.2 ± 8 for control group; *p* = 0.004 mean ± SD; **Figure 1A**) and is able to reduce neurological deficit on day 7 post-ischemia.

This neuroprotective effect may be due to the reduction of the proinflammatory cytokines TNF-α and IL-1β and the increase of IL-4, as was observed by the Manguin group in a model of ischemia in diabetic mice [75]. Additionally, the production of neurotrophic factors—known to be implicated in the processes of neural survival and proliferation of neuron precursor cells—could also be involved [76].

On the other hand, recovery from neurological deficit can be achieved by diverse mechanisms; for instance, neuroprotection exerted by Cop-1 could be limiting tissue damage caused by inflammation, this could allow the proper functioning of remaining neuronal connections. Functional recovery could also be the result of neurogenesis induced by Cop-1. Neurogenesis is a phenomenon that can replace neurons that died during the ischemic insult by allowing the substitution of neuronal circuits and thus neurorestoration.

Our study provided evidences about the neuroprotective effect of Cop-1; however, the fact that Cop-1-induced T cells are able to produce neurotrophic factors, led us to think that, it was imperative to investigate if behind the clinical recovery there was also a possible neurogenesis phenomenon.

In the following study, we evaluated whether Cop-1 induces neurogenesis in the two neurogenic niches of the adult brain: in the subventricular (SVZ) and the

#### **Figure 1.**

*Neuroprotective effect of the copolymer-1. (A) Infarction size reduction. (B) Effect of the copolymer-1 on the neurological deficit. n = 8. Each bar represents mean ± SEM. Two-way repeated measure ANOVA. Sidak's post hoc multiple comparisons test. \*p < 0.05; \*\*p < 0.0.*

**149**

**Figure 2.**

*Neuroprotective and Neurorestorative Properties of Copolymer-1: Its Immunomodulating Effects…*

subgranular zone of the dentate hippocampus gyrus (SGZ) [77]. To accomplish the evaluation, we performed an immunofluorescence technique using a double marking of 5-bromo-2′-deoxyuridine (BrdU) and doublecortin (Dcx) at 7 and 60 days

The results were very encouraging, the neurological recovery caused by Cop-1 was observed from day 7 post-ischemia as in the first experiment [78] and was improving even in the chronic phase at 60 days (**Figure 1B**). The number of neuroblasts in the groups treated with Cop-1 was significantly higher in the two neurogenic niches at both 7 and 60 days in the SVZ and SGZ (**Figure 2**). This neurogenic phenomenon correlated with the clinical recovery of treated rats. Simultaneously, an important increase of NT-3 was observed in the area of the ischemic penumbra [79]. Cop-1-induced neurogenesis has been evaluated in other animal models such as EAE [47], Alzheimer [66], and recently in the model of permanent cerebral ischemia in diabetic male mice C57Bl6 [75]. Regarding the latter, it is important to mention that, in a previous experiment carried out by the same group, they did not observe improvement in the neurological function nor reduction in the volume of the infarction. These findings could be the result of the use of inappropriate evaluation techniques [80] as in their most recent study, they observed a reduction in infarct size of up to 30–40% and an increase in neurogenesis 7 days after permanent ischemia in the SVZ. In addition, they found a reduction of proinflammatory cytokines such as TNF-α, IL-1β, and a significant increase of IL-4 and IL-10 [75]. Neurogenesis is a mechanism widely regulated by signals that stimulate the stem cells of neurogenic niches [81]; many of these signals are produced by the choroid plexus (CP), which is a complex structure of cells considered an interface that mediates communication between the immune system and the cerebral parenchyma [82]. Therefore, trying to analyze the mechanism by which Cop-1 induces neurogenesis, we evaluate whether Cop-1 modifies the microenvironment of CP, 14 days after tMCAO. In the third investigation, we evaluated neurological recovery—which was observed according to our previous experiments [78, 79], neurogenesis and the expression of proinflammatory (IL-1β, TNF-α, INF-ϒ, and IL-17) and anti-inflammatory cytokines (IL-4 and IL-10) as well as the concentration of growth factors

In this experiment, we again proved a significant increase of neurogenesis in the groups treated with Cop-1 in both, the SVZ and SGZ [83]. This data was similar to that previously reported [79]. As for the expression of proinflammatory cytokines, we only found significant differences in the expression of IL-17, which was observed reduced in the groups treated with Cop-1. With respect to anti-inflammatory cytokines, only IL-10 was significantly increased. In this investigation, we also found a significant increase of growth factors (BDNF, NT-3, and IGF-1) in the CP [83].

*Effect of copolymer-1 on neurogenesis at 7, 14, and 60 days. (A) Neurogenesis in SVZ at 7 and 60 days. (B) Neurogenesis in SGZ at 7 and 60 days. (C) Neurogenesis in SVZ and SGZ at 14 days. n = 8 in A and B. n = 5 in C. Each bar represents mean ± SEM. Two-tailed Mann-Whitney U test. Dunn's post hoc multiple comparison test. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. SVZ: Subventricular zone and SGZ: Subgranular zone.*

*DOI: http://dx.doi.org/10.5772/intechopen.91343*

(BDNF, NT-3 and IGF-1) at the CP (**Figure 3**).

after ischemia.

*Neuroprotective and Neurorestorative Properties of Copolymer-1: Its Immunomodulating Effects… DOI: http://dx.doi.org/10.5772/intechopen.91343*

subgranular zone of the dentate hippocampus gyrus (SGZ) [77]. To accomplish the evaluation, we performed an immunofluorescence technique using a double marking of 5-bromo-2′-deoxyuridine (BrdU) and doublecortin (Dcx) at 7 and 60 days after ischemia.

The results were very encouraging, the neurological recovery caused by Cop-1 was observed from day 7 post-ischemia as in the first experiment [78] and was improving even in the chronic phase at 60 days (**Figure 1B**). The number of neuroblasts in the groups treated with Cop-1 was significantly higher in the two neurogenic niches at both 7 and 60 days in the SVZ and SGZ (**Figure 2**). This neurogenic phenomenon correlated with the clinical recovery of treated rats. Simultaneously, an important increase of NT-3 was observed in the area of the ischemic penumbra [79].

Cop-1-induced neurogenesis has been evaluated in other animal models such as EAE [47], Alzheimer [66], and recently in the model of permanent cerebral ischemia in diabetic male mice C57Bl6 [75]. Regarding the latter, it is important to mention that, in a previous experiment carried out by the same group, they did not observe improvement in the neurological function nor reduction in the volume of the infarction. These findings could be the result of the use of inappropriate evaluation techniques [80] as in their most recent study, they observed a reduction in infarct size of up to 30–40% and an increase in neurogenesis 7 days after permanent ischemia in the SVZ. In addition, they found a reduction of proinflammatory cytokines such as TNF-α, IL-1β, and a significant increase of IL-4 and IL-10 [75].

Neurogenesis is a mechanism widely regulated by signals that stimulate the stem cells of neurogenic niches [81]; many of these signals are produced by the choroid plexus (CP), which is a complex structure of cells considered an interface that mediates communication between the immune system and the cerebral parenchyma [82]. Therefore, trying to analyze the mechanism by which Cop-1 induces neurogenesis, we evaluate whether Cop-1 modifies the microenvironment of CP, 14 days after tMCAO.

In the third investigation, we evaluated neurological recovery—which was observed according to our previous experiments [78, 79], neurogenesis and the expression of proinflammatory (IL-1β, TNF-α, INF-ϒ, and IL-17) and anti-inflammatory cytokines (IL-4 and IL-10) as well as the concentration of growth factors (BDNF, NT-3 and IGF-1) at the CP (**Figure 3**).

In this experiment, we again proved a significant increase of neurogenesis in the groups treated with Cop-1 in both, the SVZ and SGZ [83]. This data was similar to that previously reported [79]. As for the expression of proinflammatory cytokines, we only found significant differences in the expression of IL-17, which was observed reduced in the groups treated with Cop-1. With respect to anti-inflammatory cytokines, only IL-10 was significantly increased. In this investigation, we also found a significant increase of growth factors (BDNF, NT-3, and IGF-1) in the CP [83].

#### **Figure 2.**

*Neuroprotection - New Approaches and Prospects*

**5. Effect of copolymer-1 on stroke**

nal circuits and thus neurorestoration.

*hoc multiple comparisons test. \*p < 0.05; \*\*p < 0.0.*

there was also a possible neurogenesis phenomenon.

The ability of Cop-1 to modify the proinflammatory milieu and to stimulate the production of growth factors encourages the idea of testing this compound on other pathologies with characteristics of secondary degeneration caused by inflammation. In line with this, the use of Cop-1 after stroke envisions an optimistic result.

As copolymer-1 has been shown to have beneficial effects in various models where neuroinflammation is a detrimental determinant, our group decided to evaluate its neuroprotective effect on cerebral ischemia. For this purpose, we used the median transient cerebral artery obstruction (tMCAO) model. Sprague-Dawley male rats were used. After being subjected to ischemia for 90 min, the rats were

In the first study, the animals were evaluated for neurological deficit at day 1 and

day 7 post-ischemia using the Zea Longa scale [74]. Then, a histological analysis was performed using hematoxylin and eosin staining to determine neuroprotection. The results indicated that Cop-1 is able to avoid up to 85.1% increase in infarct size (4.8 ± 1.5 for Cop-1 vs. 32.2 ± 8 for control group; *p* = 0.004 mean ± SD; **Figure 1A**)

This neuroprotective effect may be due to the reduction of the proinflammatory cytokines TNF-α and IL-1β and the increase of IL-4, as was observed by the Manguin group in a model of ischemia in diabetic mice [75]. Additionally, the production of neurotrophic factors—known to be implicated in the processes of neural survival and proliferation of neuron precursor cells—could also be involved [76]. On the other hand, recovery from neurological deficit can be achieved by diverse

mechanisms; for instance, neuroprotection exerted by Cop-1 could be limiting tissue damage caused by inflammation, this could allow the proper functioning of remaining neuronal connections. Functional recovery could also be the result of neurogenesis induced by Cop-1. Neurogenesis is a phenomenon that can replace neurons that died during the ischemic insult by allowing the substitution of neuro-

Our study provided evidences about the neuroprotective effect of Cop-1; however, the fact that Cop-1-induced T cells are able to produce neurotrophic factors, led us to think that, it was imperative to investigate if behind the clinical recovery

In the following study, we evaluated whether Cop-1 induces neurogenesis in the two neurogenic niches of the adult brain: in the subventricular (SVZ) and the

*Neuroprotective effect of the copolymer-1. (A) Infarction size reduction. (B) Effect of the copolymer-1 on the neurological deficit. n = 8. Each bar represents mean ± SEM. Two-way repeated measure ANOVA. Sidak's post* 

immunized in the interscapular region with a dose of 200 μg of Cop-1.

and is able to reduce neurological deficit on day 7 post-ischemia.

**148**

**Figure 1.**

*Effect of copolymer-1 on neurogenesis at 7, 14, and 60 days. (A) Neurogenesis in SVZ at 7 and 60 days. (B) Neurogenesis in SGZ at 7 and 60 days. (C) Neurogenesis in SVZ and SGZ at 14 days. n = 8 in A and B. n = 5 in C. Each bar represents mean ± SEM. Two-tailed Mann-Whitney U test. Dunn's post hoc multiple comparison test. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. SVZ: Subventricular zone and SGZ: Subgranular zone.*

**Figure 3.**

*Effect of the copolymer-1 on the expression of growth factors and IL-10. Gene expression of: (A) BDNF; (B) NT-3; (C) IGF-1; and (D) IL-10. Bars represent mean ± SEM of 5 rats from each group. \*p < 0.05, \*\*p < 0.001. Mann-Whitney U test. Dunn's post hoc multiple comparison test.*

Both growth factors and IL-10 have been reported to be directly involved in the stimulation of SVZ and SGZ stem cells; specifically, IL-10 has been observed to induce stem cell proliferation but not differentiation in primary cultures [84]. Moreover, IL-10 has immunomodulatory capacity as it inhibits the synthesis and release of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β that are known to affect neurogenesis [85]. Moreover, growth factors such as NT-3 maintain viable stem cells from neurogenic niches facilitating plasticity [86]. BDNF promotes the proliferation and survival of neuroblasts [87] and IGF-1 promotes stem cell differentiation and migration of neuroblasts [88]. Therefore, this investigation allowed to demonstrate that Cop-1 is capable of raising the expression of IL-10 and growth factors, which have beneficial effects on neurogenesis.

In order to know if Cop-1 modulates the number of leukocytes in CP and to know if these intervene in the synthesis and release of growth factors and IL-10, we evaluated the cell types present in the cerebrospinal fluid in animals submitted to tMCAO and Cop-1 therapy. The results showed a significant increment in CD8+ T cells, which positively linked with the increase in growth factors and IL-10 [unpublished data].

The increase in CD8+ T lymphocytes has been observed as an effect of copaxone immunization in patients with MS [46]. In addition, experiments performed in the EAE model have considered these cells indispensable for the development of the beneficial effect of Cop-1 [89]. However, it is necessary to identify the nature of these cells and whether the type of CD8 T lymphocytes is of a regulatory phenotype.

Finally, the combination of Cop-1 with other strategies like polyunsaturated fatty acids has shown optimistic results as together, they have a greater capacity to significantly reduce the size of the infarction in the tMCAO model [unpublished data].

**151**

**Author details**

José Juan Antonio Ibarra Arias\*

provided the original work is properly cited.

María Yolanda Cruz Martínez, Melanie Tessa Saavedra Navarrete and

Salud, Universidad Anáhuac México, Estado de México, Mexico

\*Address all correspondence to: jose.ibarra@anahuac.mx

Centro de Investigación en Ciencias de la Salud (CICSA), Facultad de Ciencias de la

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Neuroprotective and Neurorestorative Properties of Copolymer-1: Its Immunomodulating Effects…*

The existing evidence of the effect of Cop-1 in the tMCAO model has been very encouraging, as it shows a significant neurological recovery. This beneficial effect could be caused by modulatory mechanisms that allow the increase of IL-4 and the reduction of TNF-α and IL-1β at the lesion site, promoting then neuroprotection. Additionally, neurological recovery could also be reinforced by the changes induced by Cop-1 at the CP as the increase of IL-10 and growth factors in this site stimulate neurogenesis after ischemia. We consider that more investigations are needed in order to analyze in greater detail the mechanism by which Cop-1 acts so that in the medium term, it may be considered as a pharmacological alternative for patients

*DOI: http://dx.doi.org/10.5772/intechopen.91343*

suffering from a cerebrovascular event.

**6. Conclusion**

*Neuroprotective and Neurorestorative Properties of Copolymer-1: Its Immunomodulating Effects… DOI: http://dx.doi.org/10.5772/intechopen.91343*
