**2.6 Statistical analysis**

Two-way ANOVA was used to analyze behavioral and cellular data. Group differences were considered statistically significant at P < 0.05. When appropriate,

*Melatonin Pretreatment Effect in a Parkinson Disease Experimental Model Induced… DOI: http://dx.doi.org/10.5772/intechopen.106001*

*posthoc* comparisons were made with the Tukey test. All analyses were conducted with GraphPad Prism 9 Software for Mac.

#### **3. Results**

After 5 months of Mn inhalation, neither clinical alterations nor significant weight changes were detected in the exposed animals compared with controls.

The motor impairment induced by inhaling MnCl2/Mn(OAc)3 mixture was bilateral and progressive. Melatonin pretreatment showed significant results regarding protection against motor alterations and neuronal degeneration.

#### **3.1 Motor behavior**

When evaluating the motor deficit that appeared in the beam-walking test and the single pellet reaching task after 5 months, we observed that the melatoninpretreated/Mn-exposed group obtained better coordination performance in both cases while the exposed group progressively decreased their motor skills (**Figures 3** and **4**). **Figure 3** depicts the beam-walking test results. The average time taken to cross it was determined; after 14 inhalations, the mice in the Mn mixture-exposed group showed an increase in the time to perform the test, and the melatonin-pretreated/Mn-exposed and control groups remained constant. We found significant differences between melatonin-pretreated/Mn-exposed and Mn-exposed/no treatment groups since the latter had greater difficulty crossing the beam.

Regarding the single pellet reaching task, in **Figure 4** we can observe that during the 5 months, the control group maintained an average of 16 correct answers, while in the Mn-exposed/no treatment group, after the fourth inhalation, the number of successes decreased and dropped to an average of 5; however, the melatonin-pretreated/ Mn-exposed group had significant differences compared to the Mn-exposed/no treatment group; it was clear that the motor coordination on melatonin-pretreated mice was not so affected.

Motor qualitative assessment after Mn mixture inhalation/no treatment resulted in tremor and bradykinesia, postural shifts and limb extension impairments (resulting in evident shortened reaches), paw aim, and supinationpronation during grasping and pellet release into the snout (**Figure 5** sequence **B**). Mn-exposed/no treatment mice exhibited abnormal movements when recollecting the pellet after Mn-exposure. The paw is often fully pronated and moves either, laterally over the pellet, or the mouse slaps at the pellet from above. These mice repeatedly could not accurately close their fingers around the pellet and took it to the slot without lifting the paw. Mice also failed to supinate the paw entirely and placed their snout into the slot to retrieve the pellet with the tongue. When the paw was pulled out through the slot, Mn-exposed mice frequently twisted their bodies and "chased" the pellet with the mouth instead of opening the fingers and placing it into the snout. However, when observing the control and the melatonin-pretreated/Mn-exposed groups, those animals advanced their forelimb through the slot and extended their fingers, supinated their paw to present the food to the mouth, and extended their digits to release the food into the snout (see **Figure 5** sequences **A, C**).

**Figure 3.**

*Mean of the time traveled on the beam ± SE (\* Mn-exposed groups vs. control group, P < 0.05; # = melatoninpretreatment vs. Mn-no treatment exposed group; two-way ANOVA followed by Tukey's posthoc test).*

#### **Figure 4.**

*Reaching success (number of pellets obtained out of 20; mean ± S.E.) by control, melatonin-pretreated/ Mn-exposed, and Mn-exposed/no treatment mice in the single-pellet reaching task. Note that the Mn-exposed/no treatment group is impaired after four inhalations (\* P < 0.001 vs. control group; # P < 0.001 between melatoninpretreated/Mn-exposed and Mn-exposed/no treatment groups. ANOVA with post hoc test).*

*Melatonin Pretreatment Effect in a Parkinson Disease Experimental Model Induced… DOI: http://dx.doi.org/10.5772/intechopen.106001*

#### **Figure 5.**

*Representative still frames of the three groups. The control (sequence A) and the melatonin-pretreated/ Mn-exposed (sequence C) animals advanced their forelimb through the slot and extended their digits, and also supinated their paw to present the food to the mouth and extended their fingers to release the food into the mouth. In contrast, the Mn mixture-exposed/no treatment mice (sequence B) showed impairments using extreme postural adjustments advancing the limb diagonally through the slot, making many short attempts rather than aligning the limb with the midline of the body. The fingers are concurrently adducted. The paw comes in from the side or slaps laterally, and digits do not contact the food pellet.*

#### **3.2 Dendritic spines**

When analyzing the number of dendritic spines of the striatal MSN with the Golgi impregnation technique, it was observed (**Figures 6** and **7**) that the number of dendritic spines significantly decreased in the Mn-exposed/no treatment group and in the melatonin-pretreated/Mn-exposed group compared to control; however, it is also shown that the melatonin-pretreated/Mn-exposed group is statistically different from the Mn-only exposed group, in other words, despite having a significant loss of dendritic spines, they lost fewer spines than the group that did not receive treatment.

#### **3.3 TH+ immunocytochemistry**

Regarding the number of SNc dopaminergic neurons (TH+ ) (**Figures 8** and **9**), it was observed that the Mn-mixture-exposed/no treatment group presented a significant loss of neurons compared to the melatonin-pretreated/Mn exposed group; both groups had significant differences compared to the control group.

#### **Figure 6.**

*Golgi-stain analysis. Striatal MSN dendritic spines density of the control, melatonin-pretreated/Mn-exposed, and Mn-no treatment groups (\* = versus control group, P < 0.05; # = melatonin-pretreatment vs. Mn-no treatment exposed group; two-way ANOVA followed by Tukey's posthoc test).*

#### **Figure 7.**

*Dendritic spine density. Photomicrographs of representative Golgi-stained MSN of the striatum from control (A, a), melatonin-pretreated/Mn-exposed (B, b), and Mn mixture-exposed/no treatment groups (C, c). Both Mn-exposed groups had a significant decrease in the total number of spines. However, melatonin-pretreated/ Mn-exposed group showed less dendritic spine loss and an almost a well-preserved dendritic spines density (magnifications: A, B, C 10×; a, b, c 100×).*

*Melatonin Pretreatment Effect in a Parkinson Disease Experimental Model Induced… DOI: http://dx.doi.org/10.5772/intechopen.106001*

#### **Figure 8.**

*SNc TH+ cell count. The data are presented as mean ± SEM. It is observed that both Mn-exposed groups showed a drastic decrease in cells. It is important to note that animals receiving melatonin pretreatment before Mn inhalation lose fewer neurons than Mn-exposed/no treatment group. (\* = P < 0.05 versus control group; # = P < 0.05 melatonin-pretreated/Mn exposed group vs. Mn-exposed/no treatment group, ANOVA test followed by Tukey's posthoc test).*

#### **Figure 9.**

*Representative TH-immunostained from coronal sections containing the SNc of A. Control group; B. Melatoninpretreated/Mn exposed group, and C. Mn-exposed/no treatment group. Note the great cell loss in the Mn-exposed/ no treatment group; although there was a neuronal loss in the pretreated group, it was less drastic than in the untreated-Mn exposed group (magnification 10×).*

However, it is observed that, although the melatonin-pretreated/Mn exposed group had a significant loss of TH+ neurons, this loss was less than the group that only inhaled Mn, with significant differences between the two groups.

### **4. Discussion**

PD is a degenerative disorder, determined clinically from movement alterations, and it is reported that the motor symptoms appear relatively late when 80−90% of SNc dopaminergic neurons have been lost [4, 5, 9, 68]. Progressive cell loss leads to increased physical disability, followed by a cognitive decline [1]. Ordoñez-Librado and collaborators [27] indicate that the MnCl2/Mn(OAc)3 inhalation model is an alternative that allows us to carry out evaluations in the different stages of evolution of the disease since it produces progressive and bilateral degeneration of the SNc dopaminergic neurons in exposed mice, as well as motor alterations, for which it is the model most closely related to what happens in humans [28].

#### **4.1 Motor behavior**

Our results showed that in both motor tests, the mice pretreated with melatonin did not have such a drastic decrease in motor coordination as observed in the animals exposed to Mn with no treatment. In previous works from our group, Sánchez-Betancourt et al. [28] and Ordoñez-Librado et al. [26] reported that animals exposed to the mixture of Mn compounds present motor alterations as the number of inhalations increases. Motor alterations are closely related to basal ganglia, which have a fundamental role in the initiation and execution of continuous movement; in other words, they participate in the planning of complex movements [69], for example, in the automatic control of movements such as gait primarily through its interaction with cortical motor areas. However, disruption of this system can lead to gait disturbances as in PD [70]; gait disturbances are common symptoms of parkinsonism [71, 72]; PD patients have a shortened stride length with a shuffling step and reduced speed (festinant) [73]. Our results also agreed with Fernagut et al. [74], who observed that MPTP-lesioned mice presented alterations in the extremities' coordination. In the beam walking test results, the mice exposed to Mn/no treatment did not coordinate their limbs correctly and had great difficulty climbing the beam. This group also manifested akinesia. It is well known that akinesia rapidly becomes intolerable when PD patients are not LD-treated [30]. This symptom was not present in the melatonin-pretreated/Mn exposed animals. According to this, melatonin bioavailability in the brain is observed from the first 30 min after oral administration. It continues to exert its antioxidant properties through its metabolites for extended periods [75, 76], thus facilitating the reduction of abnormal motor behavior.

On the other hand, in the single pellet reaching task, which consists of a series of motor subcomponents [77], since reaching movements are shortened and limb pronation and supination are impaired due to the decrease in dopamine [78] in Mn-exposed mice [27], in humans, we can observe that manual dexterity worsens as PD progresses [79]; Farr and Whishaw [65] mention that rodents reaching movements are very similar to those of humans and, due to this, homology between them is suggested. Whishaw et al. [78] indicated that using this test is helpful in studies of PD subjects to assess movement efficacy and evaluate

*Melatonin Pretreatment Effect in a Parkinson Disease Experimental Model Induced… DOI: http://dx.doi.org/10.5772/intechopen.106001*

dopamine denervation. Conversely, regarding the melatonin-pretreated/Mn exposed group, we observed less deterioration even though they inhaled the mixture of Mn compounds. The motor behavior of these animals was very similar to those of the control group. It has been reported that melatonin systemic administration protects SNc dopaminergic neurons against 6-OHDA neurotoxicity in the rat [52, 80]. The effect is accompanied by a significant motor behavior recovery. In this regard, Singh et al. [52] have reported that melatonin pretreated animals and subsequently 6-OHDA-lesioned and treated with melatonin for seven more days showed a diminution in the number of apomorphine-induced rotations, improved posture, and slowness of movement compared to 6-OHDA-lesioned treated with vehicle solution group.

It has also been reported that elevated ROS participate in Mn-neurotoxicity [81–84]; this has been evidenced by the reduction in brain GSH levels and the loss of SOD activity [83]. Melatonin stimulates antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione reductase (GRd) [76, 85]. These results demonstrate that melatonin might have beneficial effects on PD treatment.

#### **4.2 Dendritic spines**

We found a significant dendritic spine loss in both Mn-exposed groups, being more drastic in the Mn-exposed/no treatment group. Our findings agreed with what was reported previously by our group [26, 28]. We found a decrease in the density of dendritic spines after Mn mixture exposure. Also, it has been reported that striatal MSN of *postmortem* PD brains and the brains of PD animal models in rodents and primates show severe spine loss [86–88]. In this regard, Archibald and Tyree [89] suggested that Mn interacts with dopaminergic catechol groups causing dopamine depletion and damage to these neurons; such loss of striatal dopamine is therefore associated with the reduction of MSN dendritic spines as a compensatory mechanism [90] since by reducing the number of dendritic spines, it also decreases the possibility of glutamatergic synaptic contacts [91–93], avoiding death due to excitotoxicity [92, 93] because cortex excitatory innervation [94].

However, we observed that melatonin pretreatment has a protective effect on the nigrostriatal dopaminergic pathway since, as mentioned, the pretreated animals showed better stability and coordination in the motor tests. We can also observe that in the group pretreated with melatonin, the mice had a significant loss of dendritic spines compared to the exposed group. This agrees with what was reported by Anaya-Martinez et al. [80], where they showed that the animals treated with melatonin that were lesioned with 6-OHDA showed improvement at 28 days after the lesion, as well as the administration of melatonin prevented the loss of MSN dendritic spines, suggesting that melatonin can activate some signaling pathways to increase the defense against ROS. Melatonin stimulates the system of antioxidant enzymes [76], such as SOD, GPx, GRd, and catalase [95], preventing lipid peroxidation in the striatum, and preserving a greater number of dopaminergic neurons in the SNc; this can be explained by the free radical scavenging effect of melatonin and some of its metabolites [76]. Therefore, it is likely that due to these properties, melatonin prevents dopaminergic neurons from degenerating, which is evidenced by the preservation of dendritic spines, since by avoiding the loss of TH<sup>+</sup> neurons, dopaminergic transmission to the striatum is maintained, as well as the dendritic spines integrity [96].

#### **4.3 TH+ immunocytochemistry**

We observed an intense decrease of TH+ cells after Mn inhalation in both exposed groups. Our results coincide with Damier et al. [97], who found that PD patients displayed a dopaminergic neurons decrease of up to 95% depending on the time of clinical evolution. Likewise, it has been demonstrated that the medial forebrain bundle unilateral 6-OHDA lesion reduces 98% of the SNc ipsilateral number of TH- immunoreactive neurons [98, 99]. Some studies have found that Mn exposure causes a decrease in the number of SNc dopaminergic neurons since the Mn enters them through the dopamine transporter (DAT) [27, 28, 100, 101], and intracellularly Mn accumulates in the mitochondria via the 2+ uniport channel [102], inhibiting respiratory chain complex I and thus, promoting the ROS formation [83], leading neurons to oxidative stress and therefore dead. It is known that melatonin increases complex I and IV mitochondrial activity by raising mitochondrial DNA expression [103]. In addition, melatonin's free radical scavenger property neutralizes radicals such as OH• and O2 •− [58]. Our findings showed that melatonin pretreatment partially prevents SNc dopaminergic cell death produced by Mn mixture inhalation. Among the Mn-inhalation consequences are ROS production [81–83] and the complexes I and IV inhibiting the mitochondria electron transport chain [104]. Inhibition of these complexes has also been described in PD patients SNc. This inhibition triggers energy depletion and increases mitochondria free radical concentration [105].

In the present work, for melatonin pretreated/Mn-exposed group, although the TH+ cell percentage decreases (compared to the control group), the loss was less severe than that observed in the Mn-exposed/no treated group. Melatonin antioxidant effects and its protective characteristic against the uncoupling of the electron transport chain of several toxins in the mitochondria are summarized by Acuña-Castroviejo et al. [106]. These data give rise to further analyses based on this hypothesis. *In vivo* melatonin pretreatment studies in experimental PD models are scarce. These authors also reported that melatonin prevented lipid peroxidation increase and the decrease in striatal TH+ terminals after MPTP single dose, concluding that melatonin was able to prevent the damage caused by this drug in the striatal dopaminergic axons. In this way, Ortiz et al. [107] found apoptosis of the SNc dopaminergic neurons with an MPTP-unique dose; melatonin prevented cell death. Moreover, melatonin was able to avoid the reduction in striatal TH+ immunoreactivity and the mitochondrial complex I alteration induced by 6-OHDA-lesion [108].

#### **5. Conclusion**

The results obtained in the present work provide evidence that melatonin pretreatment performs as a dopamine regulator protecting partially the striatal MSN dopaminergic denervation by preserving the dendritic spines and preventing the SNc TH+ cell death, causing motor behavior recovery, as melatonin-pretreated mice displayed better motor performance and no parkinsonian symptoms, compared to Mn-exposed/ no treatment mice.

It is likely that in the MnCl2/Mn(OAc)3 inhalation PD model, we are recreating an initial stage of the disease since the Mn-exposed mice lost ~70% of the dopaminergic cells, so we believe that it would be helpful to give melatonin to patients who start with the disease, in order to delay the symptoms and dopaminergic cell death and, above all, the start of L-DOPA treatment since it produces very disabling side effects for the patient.

*Melatonin Pretreatment Effect in a Parkinson Disease Experimental Model Induced… DOI: http://dx.doi.org/10.5772/intechopen.106001*
