**3. Vulnerability of dopaminergic neurons to 6-OHDA**

6-Hydroxydopamine (6-OHDA) is a highly oxidizable dopamine analog, which can be captured through the dopamine transporter (DAT) [25]. Till date, three mechanisms have been proposed to explain the cytotoxic effect of 6-OHDA: (1) intra- or extracellular auto-oxidation, which favors the production of hydrogen peroxide, superoxide and hydroxyl radicals [13]; (2) formation of hydrogen peroxide by the effect of monoamine oxidase [32]; and (3) direct inhibition of the mitochondrial respiratory chain I complex [33].

the progressive loss of dopaminergic neurons of the SNpc, emulating the nigrostriatal dam-

Animal Model of Parkinson Disease: Neuroinflammation and Apoptosis in the…

http://dx.doi.org/10.5772/intechopen.71271

379

Kirik and colleagues [20] described that the ventrolateral region of striatum in the rat that receives afferents from the motor and the sensorimotor areas of the cortex and exclusive innervations of the SNpc. The dorsomedial region of the striatum has a mixture of innervations of the SNpc, the VTA, the frontal cortical area and the limbic system. Therefore, 6-OHDA lesions involving the dorsomedial region have general effects on locomotion and drug-induced (such as amphetamine and apomorphine) rotational behavior, while lesions affecting the ventrolateral region show effects pronounced at the beginning of the movement, sensorimotor orientation and fine motor behavior [20]. In addition, they observed that a single dose given at one striatal site causes 80% reduction in striatal innervation, and a loss of about 90% of the nigral dopaminergic population; while the dose administered at several sites of the striatum generates damage in extra-striatal innervation [20]. The effect of intra-

age observed in PD (**Figure 1**) [23, 24, 54–56].

striatal injection depends on the site of injury and dose.

**Figure 1.** Overview of cellular processes promoted by 6-OHDA in rat.

**4.1. Intra-striatal model**

These mechanisms can act independently or in combination to generate reactive oxygen species (ROS) [30]. Injection of 6-OHDA increases iron levels in the SNpc, which further induces the generation of ROS and cytochrome c release [13]. ROS and quinones derived from 6-OHDA diminishes the antioxidant capacity of the cell, resulting in oxidative damage to proteins, lipids and DNA [34]. Miyama and colleagues observed that 6-OHDA treatment decreased cellular glutathione content in a time-dependent manner before the oxidation of DJ-1 (oxDJ-1), a PD-related endogenous protein [35]. The oxidative stress generated can be amplified by the increase of free calcium in the cytoplasm, which is the product of glutamate excitotoxicity or by the loss of mitochondrial membrane permeability [36].

The dopaminergic neurons of the SNpc are vulnerable to oxidative stress induced by 6-OHDA, because they have increased basal levels of ROS, as well as low levels of glutathione peroxidase, an enzyme that reduces hydrogen peroxide to water [37]. The dopamine neurotransmitter has a high susceptibility to auto-oxidize and to become neuromelanin, which promotes the formation of hydroxyl radicals. This when combined with iron accumulated normally at high concentrations in dopaminergic neurons [3, 38], affects its elimination capacity. Also, during the oxidation of dopamine, several transient metabolites are formed such as dopamine o-quinone, aminochrome and 5,6-indolequinone [39]. These metabolites induce the formation of superoxide and adducts with several proteins like parkin [40, 41], tyrosine hydroxylase (TH) [42], glutathione peroxidase 4 [43] and several others. Indeed, it has been proposed that 5,6-indolequinone is the most reactive species that could form adducts with alpha-synuclein generating neurotoxic oligomers [7].

However, not all dopaminergic neurons of SNpc are vulnerable to 6-OHDA toxicity because there are subpopulations of dopaminergic neurons in SNpc expressing calcium-binding proteins such as calretinin and calbindin-D28k, which prevent the accumulation of intracellular calcium, avoiding the consequent excitotoxicity due to glutamate, and the cytotoxic action of 6-OHDA [44, 45]. The redox system plays an important role in protecting the dopaminergic neurons against oxidative stress. The thioredoxin and glutaredoxin systems directly mediate reduction of the 6-OHDA-quinone *in vitro* and protect neurons against dopamine-induced cell death [46].

## **4. 6-OHDA model**

Ungerstedt and colleagues demonstrated that intracerebral stereotaxic injection of 6-OHDA causes degeneration of the nigrostriatal pathway [10]. To evaluate the 6-OHDA toxicity *in vivo*, three models of injury have been developed: (1) the medial forebrain bundle injection [47, 48], (2) the intranigral lesion [21, 49] and (3) the intra-striatal injury [20, 50–52]. Although injury to the medial forebrain bundle and the intranigral lesion is useful to demonstrate the immediate neurotoxic effects, it has the disadvantage of causing rapid and generalized degeneration of the injured nucleus [53], being unfavorable models to study the cell death type generated by long-term oxidative stress. However, the unilateral or bilateral intra-striatal model does cause the progressive loss of dopaminergic neurons of the SNpc, emulating the nigrostriatal damage observed in PD (**Figure 1**) [23, 24, 54–56].

#### **4.1. Intra-striatal model**

which favors the production of hydrogen peroxide, superoxide and hydroxyl radicals [13]; (2) formation of hydrogen peroxide by the effect of monoamine oxidase [32]; and (3) direct

These mechanisms can act independently or in combination to generate reactive oxygen species (ROS) [30]. Injection of 6-OHDA increases iron levels in the SNpc, which further induces the generation of ROS and cytochrome c release [13]. ROS and quinones derived from 6-OHDA diminishes the antioxidant capacity of the cell, resulting in oxidative damage to proteins, lipids and DNA [34]. Miyama and colleagues observed that 6-OHDA treatment decreased cellular glutathione content in a time-dependent manner before the oxidation of DJ-1 (oxDJ-1), a PD-related endogenous protein [35]. The oxidative stress generated can be amplified by the increase of free calcium in the cytoplasm, which is the product of glutamate

The dopaminergic neurons of the SNpc are vulnerable to oxidative stress induced by 6-OHDA, because they have increased basal levels of ROS, as well as low levels of glutathione peroxidase, an enzyme that reduces hydrogen peroxide to water [37]. The dopamine neurotransmitter has a high susceptibility to auto-oxidize and to become neuromelanin, which promotes the formation of hydroxyl radicals. This when combined with iron accumulated normally at high concentrations in dopaminergic neurons [3, 38], affects its elimination capacity. Also, during the oxidation of dopamine, several transient metabolites are formed such as dopamine o-quinone, aminochrome and 5,6-indolequinone [39]. These metabolites induce the formation of superoxide and adducts with several proteins like parkin [40, 41], tyrosine hydroxylase (TH) [42], glutathione peroxidase 4 [43] and several others. Indeed, it has been proposed that 5,6-indolequinone is the most reactive species that could form adducts with alpha-synuclein generating neurotoxic oligomers [7].

However, not all dopaminergic neurons of SNpc are vulnerable to 6-OHDA toxicity because there are subpopulations of dopaminergic neurons in SNpc expressing calcium-binding proteins such as calretinin and calbindin-D28k, which prevent the accumulation of intracellular calcium, avoiding the consequent excitotoxicity due to glutamate, and the cytotoxic action of 6-OHDA [44, 45]. The redox system plays an important role in protecting the dopaminergic neurons against oxidative stress. The thioredoxin and glutaredoxin systems directly mediate reduction of the 6-OHDA-quinone *in vitro* and protect neurons against dopamine-induced cell death [46].

Ungerstedt and colleagues demonstrated that intracerebral stereotaxic injection of 6-OHDA causes degeneration of the nigrostriatal pathway [10]. To evaluate the 6-OHDA toxicity *in vivo*, three models of injury have been developed: (1) the medial forebrain bundle injection [47, 48], (2) the intranigral lesion [21, 49] and (3) the intra-striatal injury [20, 50–52]. Although injury to the medial forebrain bundle and the intranigral lesion is useful to demonstrate the immediate neurotoxic effects, it has the disadvantage of causing rapid and generalized degeneration of the injured nucleus [53], being unfavorable models to study the cell death type generated by long-term oxidative stress. However, the unilateral or bilateral intra-striatal model does cause

inhibition of the mitochondrial respiratory chain I complex [33].

378 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

excitotoxicity or by the loss of mitochondrial membrane permeability [36].

**4. 6-OHDA model**

Kirik and colleagues [20] described that the ventrolateral region of striatum in the rat that receives afferents from the motor and the sensorimotor areas of the cortex and exclusive innervations of the SNpc. The dorsomedial region of the striatum has a mixture of innervations of the SNpc, the VTA, the frontal cortical area and the limbic system. Therefore, 6-OHDA lesions involving the dorsomedial region have general effects on locomotion and drug-induced (such as amphetamine and apomorphine) rotational behavior, while lesions affecting the ventrolateral region show effects pronounced at the beginning of the movement, sensorimotor orientation and fine motor behavior [20]. In addition, they observed that a single dose given at one striatal site causes 80% reduction in striatal innervation, and a loss of about 90% of the nigral dopaminergic population; while the dose administered at several sites of the striatum generates damage in extra-striatal innervation [20]. The effect of intrastriatal injection depends on the site of injury and dose.

**Figure 1.** Overview of cellular processes promoted by 6-OHDA in rat.

The intra-striatal injection of 6-OHDA mainly affects dopaminergic neurons of the SNpc, and it also generates a reduction of dopaminergic neurons in the VTA, which form the mesolimbic pathway and innervate to the nucleus accumbens [28, 57]. The loss of dopaminergic neurons in the VTA does not exceed 20% of the population, and the damage does not progress over time, as observed in the SNpc. The 6-OHDA model does not replicate the presence of Lewy bodies [8], and for this reason, murine models with alpha-synuclein have been established. These approaches are based on gene knockout models [58], or gene overexpression [59] and intracerebral injection of alpha-synuclein [60]. These approaches might be the relevant in understanding the degeneration of the nigrostriatal pathway and its impact on other brain nuclei, but further research is still needed.

cell damage. Another body of evidence suggests that the increase in the activation of glial cells, and the consequent release of pro- and anti-inflammatory cytokines at the site of damage, could increase the cytotoxicity of 6-OHDA [26]. Overexpression of human alpha-synuclein in a mouse model of PD showed enhanced expression of proinflammatory cytokines and microglial activation [81]. Recently, the studies focused on NG2 cells, mitochondrial dysfunction or Lewy body accumulation (trend topic based in alpha-synuclein model) has been relevant to understand

Animal Model of Parkinson Disease: Neuroinflammation and Apoptosis in the…

http://dx.doi.org/10.5772/intechopen.71271

381

The majority of studies indicated that apoptosis is the main type of cell death produced by 6-OHDA, but necrosis and autophagy contribute on neurodegenerative process also [29, 82, 83]. Given the variety of experimental models, it is not still possible to determine the proportion of dopaminergic neurons of the SNpc affected by one or other types of cell death. However, the convergence of several types of cell death could explain the time course of

Cell death has been highlighted as the final effect of 6-OHDA cytotoxicity. Several techniques are used to determine cell death type in dopaminergic neurons in rats (TUNEL, silver staining, and immunostaining to caspase-3, GSK-3β, Bax, Bad) [85–87]. Interestingly TUNEL technique is unspecific to identify apoptosis because on *in vitro* studies the 6-OHDA induces necrosis at same dose used *in vivo* [88, 89]. So the use of other apoptotic markers is recommended to show the loss of cellular integrity or specific chromatin condensation on the dopaminergic

Caspase-3 is the major effector caspase in neurons and its activation has been demonstrated by applying neurotoxins *in vitro* and *in vivo.* This cysteine protease is enrolled both in intrinsic and in extrinsic apoptotic pathway [90–92]. In *in vivo* studies, its presence has been evidenced 1 week after intra-striatal injection of 6-OHDA in rats [78, 93]. Most *in vivo* studies have demonstrated the expression of caspase-3 in different cell death models, suggesting that caspase-3 activation is involved in programmed cell death of the SNpc [92, 94, 95]. However, some recent studies are unable to confirm the presence of active caspase-3 or caspase-9 and, based on this, state that these caspases are not involved in the apoptosis of dopaminergic neurons of the SNpC [96, 97]. This controversy is further exacerbated by recent findings demonstrating the involvement of caspase-3 in non-apoptotic functions, such as the activation of microglia [98, 99]. Although most authors agree with the involvement of caspase-3 in the 6-OHDA-induced neurodegeneration, the doubt still remains if caspase-3 expression only leads to neuronal death. It has therefore been necessary to explore other markers of the apoptotic process and in this regard, scientists have highlighted the study and role of glycogen synthase kinase 3β (GSK-3β). GSK-3β is involved in the signaling pathway of neuronal apoptosis activated by oxidative stress [100], a central factor in the neuropathological process of PD [101]. GSK-3β is activated by phosphorylation of the tyrosine residue 216 (Y216), located in the kinase domain and inactivated by the phosphorylation of serine 9 (S9) [100]. It was observed that a single dose of 6-OHDA administered in the neostriatum of the rat causes caspase-3 and GSK-3β expression,

neuroinflammatory process and define alternative therapeutic targets for PD.

degeneration and the activation of the neuroinflammatory process [84].

**6. Apoptosis**

neurons of the SNpc [51].
