**6. Apoptosis**

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

Neuroinflammation in PD is characterized by microgliosis and astrogliosis increased around the dopaminergic neurons in SNpc [61]. These cellular process promotes high levels of expression of major histocompatibility complex type II (MHC-II) [62], chemokine receptors, integrins, neurotrophins and several other markers [63]. Elevated levels of pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), nitric oxide and reactive oxygen or nitrogen species (ROS/RNS) by NADPH oxidase system or by mitochondria are also observed in PD patients [31, 64]. Recently it has been demonstrated that copperzinc superoxide dismutase (SOD1) released by microglial cells, or a TNF receptor 2 selective

Injury of CNS leads to cell death, cellular swelling, excitotoxicity and the release of free radicals and nitric oxide, which triggers a strong glial response [67, 68] referred as reactive gliosis, involving the activation of microglia, astrocytes, oligodendrocytes and Neuron/glial 2 (NG2) cells [69, 70]. After injury, mature astrocytes proliferate and acquire stem cell properties suggesting their capacity to promote regeneration [71]. Depending on the stimulus and intensity of the lesion, all the three types of glia directs the cell either toward the neuroprotection by producing neurotrophic factors or toward the neurodegeneration by producing apoptotic mediators and ROS/RNS. However, NG2 cells, with their neurogenic [72], oligodendrogenic [73], astrogenic [74] and microgliogenic properties play indirect role in directing the cell toward apoptosis or protection. The presence of NG2-positive cells has been identified in SNpc but not in the striatum of the rat [75]. A recent study in a murine paradigm showed that conversion of NG2 cells to astrocytes to produce cerebral dopamine neurotrophic factor (CDNF) is anti-inflammatory in 6-OHDA-induced rat PD model [76]. However, studying the role, mode of activation and

agonist, could confer neuroprotection against 6-OHDA toxicity *in vivo* [65, 66].

conversion of NG2 cells could give further clues to the field of neuroinflammation.

The neuroinflammatory process has been evaluated through glial cell markers such as glial fibrillary acidic protein (GFAP) for astrocytes [77, 78] and OX-42 or Iba-1 antibodies to microglia [79, 80]. The temporal course of activation of these glial populations has been determined by the neurotoxic effect, from day 3 post-injury [51], and even its activation was observed up to 3 weeks after injury with 6-OHDA [78]. The neuroinflammatory process to that precedes the death of nigral dopaminergic neurons (2 weeks post-injury) is probably a mechanism indicating

nuclei, but further research is still needed.

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

**5. Neuroinflammation**

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

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 neurons of the SNpc [51].

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, loss of cytoskeletal integrity, TH levels decreased and activation of apoptotic process in dopaminergic neurons of SNpc [51, 85, 92].

processes activated by oxidative stress (neuroinflammation and neuronal death), and conse-

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

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

383

This work was supported by Consejo Nacional de Ciencia y Tecnología (Cátedra CONACyT # 1840 for DHB) and the Instituto de Neuroetología from Universidad Veracruzana (DGI-174332015137 to MJRH). Special thanks to Nancy Gabriela Vazquez Ixtepan, Cesar Augusto

quently a useful model to understand the mechanisms of novel therapies for PD.

Sandoval Vargas and Erick Hernandez Baltazar for the illustration resources.

**Acknowledgements**

**Abbreviations**

6-OHDA 6-hydroxydopamine

BBB blood–brain barrier

CNS central nervous system

DAT dopamine transporter

GFAP glial fibrillary acidic protein GSK-3 glycogen synthase kinase-3

iNOS inducible nitric oxide synthase

MPP+ 1-methyl-4-phenylpyridinium

NAT noradrenaline transporter

NG2 neuron/glial 2

COX2 cyclooxygenase 2

AIF apoptosis-inducing factor

BDNF brain derived neurotrophic factor

CDNF cerebral dopamine neurotrophic factor

GDNF glial cell line-derived neurotrophic factor

Iba-1 ionized calcium binding adaptor molecule 1

MHC-II major histocompatibility complex type II

MPTP 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine NADPH nicotinamide adenine dinucleotide phosphate

MANF mesencephalic astrocyte-derived neurotrophic factor

Other authors demonstrated atrophy and progressive death of dopaminergic neurons dependent on translocation to the nucleus of the inducing factor of Apoptosis-inducing factor (AIF), in which there was no activation of caspase-3 or release of cytochrome C or signs of apoptosis. These researchers further demonstrate that death induced by 6-OHDA in dopaminergic neurons is mediated by activation of AIF-dependent Bax [97]. In this work, AIF activation suggests the involvement of regulated necrosis. The controversy between dependent or independent death of caspase-3 could be explained by the dose, study model and site of injury employed. However, since most evidence includes the involvement of caspase-3 in the 6-OHDA-induced apoptotic process, studies that contradict this fact suggest that 6-OHDA could also lead to neuronal death by apoptosis (independent of caspase-3) or other cell death processes (necrosis and autophagy) *in vivo*.

All the toxin-induced PD models had scant attention when it comes to the neuroprotective or regenerative strategies. Neuropathology and studies related to the correlation between inflammation and immune cells need to pay much more attention. It is of great interest to know the stimulus by which glial cells respond to the microenvironment and how do they decide whether to release neuroprotective or apoptotic mediators. It would be of interest to know if all the activated glial cells arise from a limited number of precursor cells or if all glia have equal potential to proliferate. It is also most important to study in detail about the types of receptors which are present on glial cells that play a major role in the field of neuroinflammation.
