**2. Evaluation of CBS expression in the intact trout brain and after eye injury by the western blot analysis and ELISA immunosorbent assay**

The cystathionine β-synthase enzyme has a tetramer binding with two substrates (homocysteine and serine) and three additional ligands (the coenzyme pyrodoxal 5′-phosphate, the allosteric activator S-adenosylmethionine, and heme). An assessment of CBS content by Western blot analysis showed the presence of protein with a molecular weight of 63 kDa in all the divisions of the trout brain. The quantitative CBS content in different divisions of the intact trout brain and after the mechanical eye injury is shown in **Figure 1A**. The maximum level of CBS

**309**

animals (**Figure 1B**).

**Figure 1.**

**3. Telencephalon**

*Hydrogen Sulfide as a Factor of Neuroprotection during the Constitutive and Reparative…*

expression in the intact animals was found in the brain stem, while the minimum was in the telencephalon. The cerebellum and tectum showed a medium level of CBS expression. A significant increase in the level of CBS expression was observed in all the brain divisions after the mechanical eye injury (**Figure 1A**). According to the enzyme immunosorbent assay, it was 1.86 ± 0.03 pg./mL and 3.12 ± 0.26 pg./ mL after unilateral eye injury (UEI) (*P* < 0.001). Thus, within a week after the eye injury, the concentration of CBS increased 1.7 times compared with the control

*after UEI vs. control (intact) fish (## P < 0.01); n = 20 in each group.*

*Representation of western blots of cystathionine β-synthase content in the brain of the trout* Oncorhynchus mykiss. *(A) the single protein band corresponding to a molecular weight of 63 kDa was present in the trout cerebellum, optic tectum, telencephalon, and brainstem in the control (intact) animals and at 1 week after the optic nerve damage. (B) ELISA assay of CBS in the rainbow trout brain at 1 week after UEI vs. control (intact) rainbow trout. Student's t-test was used to determine significant differences between the trout at 1 week* 

Results of the IHC CBS-labeling in the trout telencephalon showed the presence of intensely and moderately labeled cells in the pallial and subpallial regions. CBS-labeled cells were located in the superficial periventricular and subventricular pallial layers. In deep pallial areas, the number of intensively labeled cells was elevated. The presence of a H2S-producing enzyme in the brain cells is associated with the process of neurochemical signaling and, in particular, with the activation of NMDA receptors. Activation of neurons in the brain of vertebrates leads to release of neurotransmitters, including glutamate, activating the NMDA receptors, which, in turn, leads to an increase in the astrocytic intracellular calcium and longterm potentiation [3, 18]. Thus, the presence of two levels of CBS activity in the trout telencephalon indicates the mediator/modulatory intercellular interactions,

In intact trout, when labeled with polyclonal antibodies against CBS, the CBSlabeled radial glia was detected particularly in the pallial and subpallial regions

which agrees with the previously obtained data on fish [11].

*DOI: http://dx.doi.org/10.5772/intechopen.90547*

*Hydrogen Sulfide as a Factor of Neuroprotection during the Constitutive and Reparative… DOI: http://dx.doi.org/10.5772/intechopen.90547*

#### **Figure 1.**

*Neuroprotection - New Approaches and Prospects*

cant role in reducing edema and inflammation [8].

central nervous system tissues [14].

1 week after the traumatic eye injury.

of H2S in the brain injury and/or cerebral ischemia.

completely clarified thus far. H2S, like nitric oxide (NO), is known to mediate posttranslational modification of proteins by adding additional sulfur to reactive cysteine residues. This modification, referred to as S-sulfhydration, is required to activate or inactivate many classes of proteins, including the ion channels, such as the ATP-dependent potassium channels, TRPV3, TRPV6, TRPM [6], enzymes, and the transcription factors NF-kB and Nrf2 [7]. Modulation of ion channels, as well as the inflammatory and the antioxidant transcription factors, using H2S after traumatic brain injury, can play a signifi-

Recently, the involvement of H2S in cerebral ischemia, traumatic brain injury (TBI), and decrease in reactive oxygen species in the H2S-dependent mechanisms has been studied using different models [8–10]. The use of monoclonal antibodies against cystathionine β-synthase (CBS) in immunohistochemical (IHC) detection of the H2S-producing complexes in the brain of juvenile trout showed an increase in hydrogen sulfide production in different parts of the brain and CBS induction in the radial glia cells after the damage of the optic nerve [11]. It was shown that the toxic and/or neuroprotective effects of hydrogen sulfide depended on concentration: lower concentrations play a physiological role, while very high concentrations cause cell death [12, 13]. Although hydrogen sulfide is considered a gasotransmitter, there is uncertainty about the total concentration of this volatile gas or highly active anionic particles (SH-) in both plasma and

The progress in studies of the hydrogen sulfide biology has led to a conclusion that polysulfides are more significant sources of intermediate sulfhydration of proteins than H2S [3]. The H2S reactions with many signal mediators, transcription factors, and channel proteins in neurons and glial cells are known both in vivo and in vitro [7, 10]. However, still little is known about interaction of the H2S intercellular communication and its consequences in the case of a traumatic cerebral injury. Such information is necessary to determine the cytoprotective or cytotoxic effects

The study of biology of the neural stem cells, based on animal models, is becoming increasingly important, since the processes of constitutive neurogenesis occur in many areas of the animal brain [15], providing a high reparative potential of CNS. One of such models is fish, which is characterized by a high rate of reparative processes [16]. The results of preliminary studies showed an increase in proliferative activity of cells of the trout brain after damage to optic nerve [17]. To further characterize the cellular response in the trout brain after eye injury, the hydrogen sulfide-producing enzyme, cystathionine β-synthase (CBS), was analyzed using western immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical labeling of CBS in various sections of the trout brain at

**2. Evaluation of CBS expression in the intact trout brain and after eye injury by the western blot analysis and ELISA immunosorbent assay**

The cystathionine β-synthase enzyme has a tetramer binding with two substrates (homocysteine and serine) and three additional ligands (the coenzyme pyrodoxal 5′-phosphate, the allosteric activator S-adenosylmethionine, and heme). An assessment of CBS content by Western blot analysis showed the presence of protein with a molecular weight of 63 kDa in all the divisions of the trout brain. The quantitative CBS content in different divisions of the intact trout brain and after the mechanical eye injury is shown in **Figure 1A**. The maximum level of CBS

**308**

*Representation of western blots of cystathionine β-synthase content in the brain of the trout* Oncorhynchus mykiss. *(A) the single protein band corresponding to a molecular weight of 63 kDa was present in the trout cerebellum, optic tectum, telencephalon, and brainstem in the control (intact) animals and at 1 week after the optic nerve damage. (B) ELISA assay of CBS in the rainbow trout brain at 1 week after UEI vs. control (intact) rainbow trout. Student's t-test was used to determine significant differences between the trout at 1 week after UEI vs. control (intact) fish (## P < 0.01); n = 20 in each group.*

expression in the intact animals was found in the brain stem, while the minimum was in the telencephalon. The cerebellum and tectum showed a medium level of CBS expression. A significant increase in the level of CBS expression was observed in all the brain divisions after the mechanical eye injury (**Figure 1A**). According to the enzyme immunosorbent assay, it was 1.86 ± 0.03 pg./mL and 3.12 ± 0.26 pg./ mL after unilateral eye injury (UEI) (*P* < 0.001). Thus, within a week after the eye injury, the concentration of CBS increased 1.7 times compared with the control animals (**Figure 1B**).

#### **3. Telencephalon**

Results of the IHC CBS-labeling in the trout telencephalon showed the presence of intensely and moderately labeled cells in the pallial and subpallial regions. CBS-labeled cells were located in the superficial periventricular and subventricular pallial layers. In deep pallial areas, the number of intensively labeled cells was elevated. The presence of a H2S-producing enzyme in the brain cells is associated with the process of neurochemical signaling and, in particular, with the activation of NMDA receptors. Activation of neurons in the brain of vertebrates leads to release of neurotransmitters, including glutamate, activating the NMDA receptors, which, in turn, leads to an increase in the astrocytic intracellular calcium and longterm potentiation [3, 18]. Thus, the presence of two levels of CBS activity in the trout telencephalon indicates the mediator/modulatory intercellular interactions, which agrees with the previously obtained data on fish [11].

In intact trout, when labeled with polyclonal antibodies against CBS, the CBSlabeled radial glia was detected particularly in the pallial and subpallial regions

of the telencephalon, while labeling with monoclonal antibodies did not reveal similar structures [11]. The present data suggests that in the trout telencephalon CBS may label aNSCs with a glial phenotype (radial glia). Our assumption is consistent with the results of studies of the pallial neurogenic niche in adult zebrafish containing radial glia-like aNSCs with cellular bodies lining the walls of the ventricle [19]. Studies of the hydrogen sulfide biology in the mammalian brain have shown that astrocytes and glial cells constitute the main repositories of CBS in the brain [3]. In in vitro experiments, it was found that astrocytes produce 7.57 times more H2S than the microglial cells [20]. However, in the fish brain, the detection of typical astrocytic glia gives controversial results [21, 22], and radial glia is detected frequently during attempts to identify the brain glial architectonics [23].

In the surface layer of different zones of the trout telencephalon, CBS+ cells and RG and cells of neuroepithelial type, representing a part of the constitutive matrix zones of the telencephalon, were also identified. Thus, CBS+ cells were detected in the zones of constitutive neurogenesis in the telencephalon of intact animals, which is consistent with the previously obtained data on the masu salmon and carp [24]. Studies on *D. rerio* have shown that aNSCs are associated with the ventricular system. In the fish telencephalon, aNSCs have a typical morphology of radial glial and/or neuroepithelium, which can be identified with several molecular markers of aNSCs [25, 26]. Thus, it is obvious that the CBS+ cells of the pallial and subpallial regions of the trout telencephalon are the aNSCs of the neuroepithelial and glial types. Earlier studies on trout have shown that after injury in the dorsal, medial, and lateral pallial zones, the number of the PCNA+ and HuCD+ cells significantly increases, indicating growth of proliferative and neurogenic activity in the telencephalon [17]. After the traumatic eye injury, the number of CBS+ cells increased in all areas of the telencephalon, with the exception of dorso-central zone (**Figure 2**). The number of H2S-producing cells increases in the periventricular and subventricular regions of the telencephalon, which are characterized by intensification of proliferative processes that occur after the eye injury.

#### **Figure 2.**

*Density of CBS+ cells in the telencephalon of the intact trout* Oncorhynchus mykiss *and at 1 week post-injury. The one-way analysis of variance (ANOVA) followed by the student–Newman–Keuls post hoc test was used to determine significant differences between the control trout and fish after UEI (n = 5 in each group; \* P < 0.01, \*\* P < 0.05 significant differences* vs*. the control group). Dm, Dd, Dl, and Dc are the medial, dorsal, lateral, and central parts of the dorsal telencephalic area; Vl and Vm are the lateral and medial parts of the ventral nucleus of telencephalon.*

**311**

**5. Optic tectum**

*Hydrogen Sulfide as a Factor of Neuroprotection during the Constitutive and Reparative…*

An increase in another type of CBS+ cells, which are intensively labeled, having no processes, and adjacent to large moderately labeled neurons, in the telencephalon parenchyma suggests intercellular neuron/glial or neuron/microglial interactions associated with release of H2S from intensively labeled astrocyte-like cells and/or microglia [18, 27]. In addition, after the injury, the patterns of distribution of the CBS+ radial glia cells in the telencephalon are retained, which indicates an additional production of H2S in aNSCs of the glial phenotype. Studies showed that after ischemic brain damage, the additional production of H2S is provided by sulfhydration [2]. In more recent studies, it has been shown that polysulfides are 300 times more active than H2S in the TRPA1 receptor activation [3]. Polysulfides activate NMDA receptors, which is accompanied by the H2S-dependent reduction of cysteine disulfide in extracellular domain of the receptor [3]. In this context, activation of the NMDA channels by H2S is probably a detrimental condition arising from the excitotoxicity of glutamate causing calcium influx, which, in turn, leads to

The results of experimental in vitro studies have shown that the glutamate toxicity during a traumatic injury (ischemia) is attenuated by the effect of H2S on

However, there is currently no consensus on the dual role of H2S in the glutamate toxicity. Neurons that are formed in the matrix periventricular zones of the trout telencephalon represent the immature cell forms that migrate from the periventricular to subventricular layers of the brain. Such undifferentiated cells can express an incomplete set of glutamate NMDA receptors, and, therefore, those cascade processes that trigger apoptosis in mature neurons in immature cells cannot cause death. On the other hand, it is known that H2S is metabolized by mitochondria through participation in the oxidation process of the H2S-producing enzymes [13, 31]. The stress-induced H2S production in mitochondria and a subsequent

Changes in the mitochondrial membrane potential activate caspase-3 and then become attenuated with NaHS in the neuronal cell culture, which protects neurons from apoptosis [33]. Thus, the controversial role of H2S in the mammalian brain neurons raises some questions about whether the excessive production of H2S causes death of mature neurons in the trout brain as a result of eye injury. It is likely that H2S has a protective effect on the immature telencephalic cells after the injury. What phenotype (glial or neuronal) does correspond to cells that produce H2S after UEI in the trout brain? Considering our previous evidence that the cells of these zones in the trout telencephalon are HuCD+ [17], it is fair to assume that the H2S-producing cells in the trout telencephalon can represent immature neurons. However, the detection of CBS expression in the radial glia cells indicates a glial phenotype. Thus, it can be concluded that as a result of UEI in the trout telencephalon, CBS expression is activated in both the neuronal and glial cell population.

As a result of UEI in the trout tectum, the number of the CBS+ cells increases dramatically, the appearance of the dense CBS+ cell groups in different layers of the tectum is diagnosed, and the CBS expression is induced in the RG cells (**Figure 3A**). The present results of CBS labeling with polyclonal antibodies in the tectum of older trout confirm the IHC labeling data for a younger age group

and CFTR/Cl<sup>−</sup> channels [29] and activation of the GLT1 transporters [30].

*DOI: http://dx.doi.org/10.5772/intechopen.90547*

neuronal toxicity and cell death [1, 28].

increase in the ATP production were demonstrated [32].

ATP/K<sup>+</sup>

**4. CBS in the telencephalon parenchyma**

*Hydrogen Sulfide as a Factor of Neuroprotection during the Constitutive and Reparative… DOI: http://dx.doi.org/10.5772/intechopen.90547*
