Behavioral and Cytological Differences between Two Parkinson’s Disease Experimental Models

Abstract

The knowledge about the biochemical and behavioral changes in humans with PD has allowed proposing animal models for its study; however, the results obtained so far have been heterogeneous. Recently, we established a novel PD model in rodents by manganese chloride (MnCl2) and manganese acetate (Mn (OAc)3) mixture inhalation. After inhaling, the rodents presented bilateral loss of SNc dopaminergic neurons. Later, we conclude that the alterations are of dopamine origin since L-DOPA reverted the alterations. After six months, SNc significantly reduced the number of cells, and striatal dopamine content decreased by 71%. The animals had postural instability, action tremor, and akinesia; these symptoms improved with L-DOPA, providing evidence that Mn mixture inhalation induces comparable alterations that those in PD patients. Thus, this study aimed to compare the alterations in two different PD experimental models: 6-OHDA unilateral lesion and Mn mixture inhalation through open field test, rotarod performance and the number of SNc dopaminergic neurons. The results show that the Mn-exposed animals have motor alterations and bilateral and progressive SNc neurons degeneration; in contrast, in the 6-OHDA model, the neuronal loss is unilateral and acute, demonstrating that the Mn exposure model better recreates the characteristics observed in PD patients.

1. Introduction

Parkinson’s disease (PD) is a neurodegenerative disorder with movement abnormalities, which include tremor, rigidity, akinesia, bradykinesia, masked face, and postural and gait abnormalities [1] involving the continued dopaminergic cell loss projecting from the substantia nigra compacta (SNc) to the striatum. Within neurodegenerative diseases, it is the second most frequent, resulting in motor disorders, abnormal dopamine signaling, and dopamine cell death [2, 3].

While its causes are still not fully understood, experimental models have postulated essential evidence. Based on clinical and experimental findings, PD was the first neurodegenerative disorder to be modeled and treated by neurotransmitter replacement therapy [4].

When selecting an animal PD model, one has to be considering the differences and similarities between humans and animals’ behavior, physiology, and anatomy. The prevailing models have helped understand the disease’s causes and compromised resources for new treatment approaches [5]. Nevertheless, the dopamine deafferentation simulated in animals, by the wide variety of neurotoxins or genetic manipulations, some PD models destroy the dopaminergic neurons rapidly and not progressively. In genetic PD models, the dopaminergic loss, while more progressive, is limited in amount or may not occur at all [5, 6].

The typical PD models (Table 1) induce nigrostriatal dopaminergic cell loss, frequently with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), rotenone or paraquat [4, 6, 7, 8, 9]. It is known that these models induce mitochondrial dysfunction and/or reactive oxygen species, but none completely simulate the pathology and symptoms seen in humans [7, 9]. 6-OHDA and MPTP are neurotoxins that rapidly and selectively destroy the dopaminergic neurons (within 1–3 days), while PD pathogenesis obeys a progressive progression over decades [4, 10].

Model	Characteristics	Disadvantages
6-OHDA	First established model, neuronal degeneration in 24 hours. Enters via Dopamine transporter (DAT) and inhibits the mitochondrial respiratory chain	Unilateral injection, acute model.; Unilateral damage, measurable by ipsi or contralateral rotation
MPTP	Humans and monkeys produces the same symptoms/histopathology and response to L-DOPA.; It is transformed to MPP+ in the glia and enters through the DAT.	Lower susceptibility in rodents, the administration is acute or subacute. In rodents there is recovery
Rotenone	Damage to the SNc and cytoplasmic inclusions is similar to Lewy bodies.; Inhibition of mitochondrial respiratory chain.	In neuronal selectivity, not all animals are affected.

Table 1.

Some PD experimental models.

The consequences of manganese (Mn) as a PD model have been investigated since its toxicity (known as manganism) is related to extrapyramidal motor system symptoms [11, 12, 13, 14].

For a long time, there has been some controversy about Mn-induced dopaminergic alterations; whereas some researchers reported that Mn alters dopaminergic innervation, particularly in the basal ganglia, and produces Parkinson-like disorder [14, 15, 16, 17, 18], many authors suggested that Mn neurotoxicity is unlike from PD in symptoms, pathology, and etiology [18, 19], particularly in the evident preservation of SNc dopaminergic somas [20, 21, 22, 23, 24].

We recently established an innovative PD model in mice [25, 26] and rats [27] by the inhalation of the mixture of two Mn compounds, Manganese chloride (MnCl₂) and Manganese acetate (Mn(OAc)₃). After Mn mixture inhalation, the rodents presented a significant loss of SNc dopaminergic neurons (67.58%) [26]. Later, we determine whether L-DOPA treatment improves the behavior to ensure that the alterations are of dopamine origin [27, 28]. Consequently, after six months of Mn compounds inhalation, striatal dopamine concentration decreased by 71%, and SNc showed a significant reduction in the number of TH⁺ neurons. The animals presented action tremor and postural instability, which were improved with L-DOPA—suggesting that MnCl₂/Mn (OAc)₃ mixture inhalation induces comparable symptoms and neurochemical and cellular alterations to those observed in PD patients, providing a valuable model for the study of this disease [25, 26, 27, 28]. Additionally, Mn inhalation is progressive and bilateral, making it more reliable. Thus, this study aimed to compare motor alterations in two different PD experimental models: 6-OHDA unilateral lesion (the most common used PD-experimental model [29] and MnCl₂/Mn(OAc)₃ mixture inhalation through open field test determining: walking distance (ambulation), rearing and walking speed, freezing time, rotarod performance and bradykinesia and counting the number of TH neurons in the SNc.

2. Methods

Male adult Wistar rats (starting weight 180–200 g) were placed during the recovery from the 6-OHDA surgery; two weeks after the surgery or Mn mixture inhalation, they were accommodated in groups of four with an inverted dark-light cycle (12:12 h) and fed with Purina Rat Chow and water ad libitum. Body weight was recorded daily. The experimental protocol follows the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996 and the Rules for Research in Health Matters (Mexico). We strive to limit the animals’ amount and their distress. Rotarod performance and open field were tested before the lesion or Mn inhalations as preliminary parameters (average condition) of each rat’s motor coordination, postural balance, and muscle rigidity to follow up on their performance throughout the experiment. The rats were first tested in the rotarod and trained to remain on the rod at 5 rpm and 10 rpm for 2 min, discarding those that, after three consecutive days, were unable to stay on the rod [30].

2.1 Stereotactic surgery

The rats were anesthetized with sodium pentobarbitone (35 mg/kg, intraperitoneal) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). They were injected with 4 ml of saline solution containing 8 mg of 6-OHDA (Sigma Chemical Company, Mexico) and 0.2 mg of ascorbic acid (n = 6) into the left medial forebrain bundle (MFB). For the unilateral lesion, the stereotaxic coordinates were as follows: AP=–6.1 mm anterior to the interaural line; L = 1.6 mm lateral to bregma; and V = – 8.1 mm vertical from dura according to Paxinos and Watson [31]. A sham lesion was made with the vehicle using the exact coordinates (n = 6, control group). The injections were administered over a 4-min period using a Hamilton syringe attached to a glass micropipette with a 20–50 mm tip diameter.

2.2 Manganese inhalation

Inhalations were performed as described by [25, 32]. Six rats were placed in an acrylic chamber inhaling 0.04 M MnCl₂ and 0.02 M Mn (OAc)₃ one hour thrice a week for six months. Six control rats inhaled only the vehicle—deionized water—for the same period. Inhalations were performed in closed acrylic boxes (40 cm wide/70 cm long and 25 cm high) connected to an ultra-nebulizer (Shinmed, Taiwan) with 10 l/min continuous flux. The ultra-nebulizer produces droplets in a 0.5–5 mm range. A trap for the vapor was on the contrary side with a sodium bicarbonate solution to precipitate the remaining metal. During exposures, animals were constantly monitored for respiration rate, depth, and regularity. The exposure system was continuously monitored for temperature, oxygen level, and Mn concentration [25, 32].

2.3 Motor behavior

2.3.1 Rotarod performance

The rotarod consists of a four-lane rotating rod (diameter 7.5 cm) and infrared beams to detect the moment of fall. The rat’s body was placed perpendicular to the rotating axis and the head against the direction of the rotation; the animal must move forward to stay on the rod. Each rat was tested for about 15 min between the different testing speeds, thus reducing stress and fatigue. The rats were trained twice on the rotarod at the constant rate of 5 and 10 rpm for two min during three consecutive days before the performance evaluation. In the evaluating session, the rats were placed on the rod, and their performance was tested at different constant speeds (5, 10, 15, 20, and 25 rpm) for a maximum of two min at each rate. All rats were video recorded while on the rod to assess their motor coordination and posture [30]. Control, 6-OHDA-lesioned and Mn-inhaled groups were evaluated before the experimental procedures and after three and six months.

2.3.2 Open field test

The rats’ behavior in the open field was evaluated before the experimental procedures and after three and six months. The rat was placed in the center of a square arena (80 × 80 cm) with 40 cm high, opaque black walls in a quiet, red-light-illuminated room. The motor behavior was video recorded for 20 min. The geometrical coordinates of the rat position in the arena were measured from the recorded videos to obtain the spatiotemporal sequence of the movements. The following behavioral parameters were measured: walking distance (ambulation), rearing, and walking speed. Bradykinesia was estimated by the reduction in walking speed assessed by the time taken by the rat to move from one corner of the open field to the next with relative speed. The arena was cleaned with a water/alcohol (70%) solution before every behavioral testing to avoid a possible bias due to odors and residues left by rats tested earlier [30]. All experiments were carried out from 11:00 a.m. to 3:00 p.m.

The times the rat passed through the center of the arena (central lines; Figure 1) of the open field were counted and multiplied by the distance (115 cm).

Figure 1.

Open field test. Rat trace in the center of the arena.

2.4 Tyrosine hydroxylase (TH) immunocytochemistry

After six months, for the immunohistochemical study, the rats were deeply anesthetized under pentobarbitone anesthesia (35 mg/kg, i.p.) after the last behavioral test and perfused transcardially with 120 ml of 0.9% saline, followed by 300 ml of 4% paraformaldehyde.

Coronal sections (50 μm) were acquired on a vibrating microtome containing the mesencephalon for immunocytochemistry. TH (Chemicon International Inc., Temecula, California, USA; 1: 1000) immunostaining with the ABC detection method (Vector Lab MI, USA) was performed for light microscope analysis. The analysis was carried out using a computer-assisted system (Image-Pro Plus; Media Cybernetics, Del Mar, California, USA) connected by a CCD camera to an Optiphot 2 Microscope (Nikon, Mexico). The number of TH⁺ neurons was counted in 1500 mm² from each animal’s seven SNc sections per hemisphere [25, 26, 27, 28, 32].

3. Statistical analysis

An unpaired t-test was used to analyze the number of cells. Motor performances were analyzed using repeated-measures ANOVA on mean values of motor activity per certain period, and post hoc comparisons were made with Tukey’s test. Group differences were considered statistically significant at p < 0.05. All analyses were conducted with GraphPad Prism Software Inc., Version 9 for Mac [25, 26, 27, 28].

4. Results

4.1 Rotarod performance

This test estimates motor coordination and balance. Both features are under the dorsal striatum control [33, 34].

First, we explored the deficit degree in motor coordination and balance produced by the unilateral MFB lesion six months after; we found that the fall latency (or permanence on the roll) decreased significantly from 15 rpm (p < 0.05), as seen in Figure 2. Figure 2 also shows the Mn mixture-exposed rats’ results, observing a progressive decrease in rod permanence directly proportional to the months of exposure. Between the pre-exposure stage vs. three months, an evident reduction is kept in the permanence time at revolutions 20 and 25, decreasing by 46% and 8%, respectively. However, after six months, the decrease in the rode permanence time was evident in the five evaluated revolutions (5, 10, 15, 20, and 25 rpm) due to the significant falls between 70 to 90%. Figure 2 shows the performance in the test after six months of experimental performances. The pre-groups are not shown because they were considered 100%.

Figure 2.

Rotarod Permanence time at (5, 10, 15, 20, 25 rpm) of the control, 6-OHDA-lesioned, and Mn-exposed groups after six months. It is observed that the higher the rod speed, the performance in this test was more deficient. Performance in the pre-exposure test was considered 100%. (* = P < 0.005 6-OHDA-lesion and Mn-exposure versus control group; # = P < 0.005 Mn-exposure versus 6-OHDA-lesion; repeated-measures ANOVA followed by Tukey post hoc test).

4.2 Open-field test

In addition to assessing gross motor deficit, we decided to explore the effect of the lesion and Mn-inhalation on the animals’ overall spontaneous motor behavior. Open-field testing after the 6-OHDA lesion revealed that voluntary movement and exploration decreased significantly to 44.87 ± 29.50% on average (Figure 3). As seen in the representative traces of the route (Figure 4A), the animal in its intact condition moved profusely through the open field, exploring the entire perimeter of the arena and sometimes the center, with few periods of inactivity. After three months of the unilateral MFB 6-OHDA lesion, the same animal made a much smaller movement (which is reflected in the low density of lines in the x-y axes), almost did not cross the center of the arena, and presented prolonged periods in which it stopped staying in expectation, or simply immobile in the corners (akinesia), this is observed in the route as a space between the movements on the t-axis (see the middle trace of Figure 4A). The Mn-exposed group also decreased the exploratory activity compared to the pre-exposure stage (Figure 5). The animals, before Mn inhalation, traveled the average maximum distance of 5875 cm in 20 min (this value was taken as 100%); after three months of exposure, a significant decrease was observed; on average, each animal traveled a distance of 3250 cm which is equivalent to 45% decrease (Figure 5); however, at six months the decline was more evident, corresponding to 66% covering a distance of 1985.83 cm in 20 min (Figures 4B and 5).

Figure 3.

The 6-OHDA lesion altered the ambulatory performance assessed by the distance traveled, the walking distance, and the rearing frequency, inducing bradykinesia and freezing in the first ten minutes. (One-way repeated-measures ANOVA) but it did not significantly affect bradykinesia. *P < 0.001 compared with control. The data are given as the mean ± SEM).

Figure 4.

(A) The representative traces are shown in a three-dimensional form of a rat’s path (x-y in time) in the two stages of the protocol after the 6-OHDA lesion. The number of lines is proportional to the total distance traveled. (B) Images showing the path of a rat in the pre-and after Mn exposure stages. It is evident that after Mn inhalation, and presumably by the loss of dopaminergic innervation, the animals did so attached to the walls when they wandered.

Figure 5.

MnCl₂/Mn(OAc)₃ inhalation decreased ambulatory activity measured by distance traveled, rearing frequency, and walking distance, and induced significant bradykinesia and freezing during the first ten minutes in the open field (one-way repeated-measures ANOVA). * P < 0.001 compared with control (the data are given as the mean ± SEM).

As can be seen, the inhalation of Mn, by producing bilateral dopaminergic damage, accentuates the alterations in a time-dependent manner (in both rotarod and open field tests) more clearly compared to the unilateral 6-OHDA-lesioned group. Moreover, the qualitative evaluation showed that Mn-exposed animals exhibit hind-limb weakness, poverty of spontaneous movement (akinesia), slowness of movement (bradykinesia), action tremor, and postural instability.

4.3 Tyrosine hydroxylase immunocytochemistry

TH⁺ neurons counting found that in the 6-OHDA unilaterally lesioned rats, SNc had 95.4% of dopaminergic neurons lost on the ipsilateral side than the contralateral (Figures 6 and 7). The number of TH⁺ neurons in the control group, both contra and ipsilateral SNc, remained unaffected (94 ± 1.9 and 93 ± 1.7, respectively) (Figure 6). In contrast, we found a substantial loss of TH-positive neurons in the SNc of 6-OHDA lesioned animals in both contralateral (73 ± 1.9) and ipsilateral (5 ± 1.6) SNc compared to controls as shown in Figures 6 and 7.

Figure 6.

SNc TH⁺ cell number. The data are presented as the mean ± SEM. A statistically significant decrease in TH-immunoreactive cells was detected in MnCl₂/Mn(OAc)₃-exposed group and both 6-OHDA-lesioned contralateral and ipsilateral SNc; being more drastic in the 6-OHDA ipsilateral SNc (* = P < 0.05 versus control group; ANOVA test).

Figure 7.

Illustrative TH⁺ from coronal sections containing the SNc of control, 6-OHDA, and MnCl₂/Mn(OAc)₃-exposed rats; it is evident the loss of dopaminergic neurons in both experimental groups is more drastic in the 6-OHDA ipsilateral SNc (magnification 10×).

Also, after six months of MnCl₂/Mn(OAc)₃ inhalation, a significant loss of the TH⁺ neurons in the SNc was observed (70.58%) compared to the control group (Figures 6 and 7).

Figures 6 and 7 clearly show the laterality of the neural loss in the unilaterally 6-OHDA-lesioned animals, unlike the rats that inhaled Mn, who presented significant bilateral cell loss.

5. Discussion

A reliable PD experimental model must gradually mimic the physiological and behavioral symptoms such as bradykinesia, akinesia, tremor, and muscle rigidity, allowing intervention in the early stages of the disease [5, 35, 36].

There are currently several experimental models that reproduce PD. These models were aimed to induce nigrostriatal dopaminergic depletion. Selective neurotoxins such as 6-OHDA, MPTP, paraquat, or rotenone are generally used [4, 7, 9]. These models induce neuronal death by inhibiting mitochondrial function and producing ROS. Still, none fully reproduce the symptoms and pathology seen in PD patients, in part because the activity of the neurotoxins is acute, fast, unilateral, and nonprogressive, and the response to these neurotoxins is different between species [4].

Our results evidence that the alterations between the two models are similar; however, the animals exposed to the Mn mixture had time-dependent behavioral alterations, while the animals lesioned with 6-OHDA presented alterations almost immediately, the neuronal loss is massive and mainly unilateral, so it is complicated to prove a treatment with 5% remaining dopaminergic neurons.

5.1 Rotarod performance

The rotarod is commonly used to behaviorally assess the degree of dopaminergic depletion (maximum or partial) in PD models [37]; this test also assesses akinesia and postural instability symptoms.

The exact mechanism underlying this test is the failure of the dopamine-depleted rats to remain on the rod; these deficits become increasingly apparent as the speed of rotation increases and the rats are obligated to move faster. There is also evidence of loss in the ability to apply force with the affected extremities; therefore, there is fatigue [38], and they tend to fall (fall latency). It has recently been shown that PD patients present instability and movement inaccuracy which becomes more prominent when they are asked to move faster [39]. The motor alterations became more evident at 20 and 25 rpm; after six months of dopamine depletion, motor coordination decreased significantly in the five evaluated revolutions. The dopamine-depleted animals tended to remain static before starting the test, so when the rotarod was turned on, it was easy for them to lose their balance and fall or not coordinate their steps with the movement of the rod when the revolutions increased. Similar results have also been reported by Rozas et al. [37] using the rotarod test in mice that received injections of MPTP, the animals showed a reduction in the time spent on the rod, related to the SNc dopamine loss. Subsequently, Razgado-Hernandez et al. [30] report the association between the degree of motor coordination deficit and the balance produced by 6-OHDA unilateral lesion in the MFB, where there is an 89.4% loss of TH⁺ neurons in the lesioned SNc.

Our data demonstrate that the Mn-exposed rats had more trouble staying on the rod since the dopaminergic denervation is bilateral, just like the PD patients.

5.2 Open field performance

The open field is a reliable test in rodents to assess motor activity as it measures (spontaneous motor behavior) that is an analog in PD patients when they present hypokinesia (decreased ambulation) and bradykinesia (reduced speed of movement).

Among the cardinal PD symptoms are akinesia and postural instability. Akinesia can be the most disabling symptom (along with tremors). It can be subdivided into different components such as delayed initiation of movement (prolonged reaction time (bradykinesia), inability to reach an object with continuous movement, rapid fatigue with repetitive motions, and balance disorders [35, 39]. The locomotion decrease is indicative of an altered state of motor behavior typical of parkinsonism, and the reduction obtained is similar to that reported by [40] for a unilateral 6-OHDA MFB lesion. In the 6-OHDA-lesioned rats, the walking distance appears unaffected, so there is no bradykinesia; we found some akinesia probably associated with a lack of motivation to move and explore the environment.

In contrast, regarding the animals exposed to Mn, before the inhalation, the rats maintained spontaneous exploration activity; however, when Mn-exposed, the spontaneous activity significantly decreased; this decrease was progressive; after three months, the decline was 45%, but after six months it was 76%, this response indicates that the model reproduces the symptoms of hypokinesia (decreased ambulation) which causes them to walk less distance and bradykinesia since their walking speed was altered, alterations reported previously by our group [41]. We also observed tremors, a symptom the 6-OHDA-lesioned rats did not present. Qualitative inspection indicated that Mn-exposed rats display hind-limb weakness, poverty of spontaneous movement (akinesia), slowness of movement (bradykinesia), action tremor, postural instability, and freezing behavior. About these alterations, Harischandra et al. [15] stated that mice exposed subchronically to Mn by intragastric gavage displayed hypoactivity; this alteration was associated with 50% striatal dopamine depletion; Eriksson et al. [42] found that after five months of Mn exposure the animals developed unsteady gait, subsequently action tremor and were hypoactive. The animals lost strength in both upper and lower extremities, and their paw movements were clumsy [42].

It is well known that rats with both MFB 6-OHDA lesions have postural instability and little ability to preserve equilibrium after tasks with destabilizing forces. Similarly, spontaneous movements are significantly altered [43]; this has not been reported after the unilateral 6-OHDA lesion.

Parkinsonian-like tremors have been scarce in unilaterally 6-OHDA-lesioned rats [44, 45]; nevertheless, Schallert et al. [46] have reported infrequent tremors in the paw and the wrist of rats with almost complete dopamine denervation (either bilateral or unilateral). This tremor is observed when the paw is placed off the floor in a non-weight-bearing posture [46]. It is also known that bilaterally 6-OHDA- lesioned rats show most of the PD motor symptoms. However, 6-OHDA bilateral lesion is not a typical PD model since the lesioned animals need intensive care since they present adipsia and aphagia and die a few days after the lesion [47]. Therefore, the MFB unilateral 6-OHDA lesion is the most commonly used PD model, even though it does not replicate all the PD symptoms and pathological characteristics. Likewise, the acute kind of the experimental models contrasts with the dopaminergic nigral neuron’s progressive degeneration in PD [41].

5.3 Tyrosine hydroxylase immunocytochemistry

We observed a severe decrease (95%) of TH⁺ cells after the unilateral 6-OHDA lesion in the ipsilateral SNc. The MFB 6-OHDA unilateral lesion produces a severe SNc degeneration, mainly in the ipsilateral side, within the first hours after the lesion, corroborated by apomorphine-induced circling behavior [48] and TH⁺ cell count (see Figures 6 and 7). Our results concur with Surmeier [49], who reported that PD patients have up to 95% of dopaminergic neuron reduction in the advanced stages of the disease. Similarly, it has been confirmed that the MFB unilateral lesion decreases 95–98% of the SNc ipsilateral number of TH⁺ neurons [50, 51]. We also found a significant cell loss in the contralateral SNc, as reported previously by our group [52, 53]; however, the cell loss in the contralateral SNc is not enough to simulate PD symptoms. Similarly, it has been described that the reserpine rat PD model is known to induce a substantial dopamine depletion with a rapid development of striatal dopaminergic receptors supersensitivity (within 12–24 h after reserpine) [54]. Other authors have shown that most severely lesioned parkinsonian primates subjected to chronic MPTP regimens present 70–80% nigral TH⁺ cell loss and >95% striatal dopamine depletion [55].

According to previous reports [25, 26, 27, 28, 41], we found a substantial loss of TH⁺ neurons, as shown in Figures 6 and 7, displaying a very similar pattern to that reported in the middle to advanced stages of the PD; it has been described neurochemical variations in humans and animals Mn-intoxicated involving the decrease in dopamine concentration and TH⁺ immunoreactivity in the SNc and striatum [13, 15, 16, 56, 57, 58, 59]. Hence, it has been supposed that Mn interacts with catechols specific to dopaminergic neurons to diminish them promptly, causing these cells to no longer be viable [60, 61]. However, there has been controversy about Mn-inducing SNc dopaminergic damage [18, 20, 22, 24, 62, 63, 64, 65]. According to these authors, Mn exposure leads mainly to Globus Pallidus and striatal alterations without affecting the SNc dopaminergic neurons. Our results are probably because we exposed the animals to a mixture of divalent and trivalent Mn. Divalent Mn pro-oxidant activity seems to depend on trivalent Mn trace amounts, which may enable Mn²⁺ small portion to oxidize to Mn³⁺. This relationship between both Mn compounds results in a continuous redox cycle [66]. It seems that divalent Mn does not have oxidative effects; nevertheless, the transition of Mn²⁺ to Mn³⁺ accelerates its oxidant capability, which might consequence in the production of reactive oxygen species, cell membrane damage, and lipid peroxidation [61], which, in turn, could affect catecholamines [67, 68]; therefore, the inherent transformation of divalent Mn to trivalent Mn and the existence of more trivalent Mn might provoke mitochondrial dysfunction and more reactive oxygen species [68, 69] manifested as the evident motor alterations and the dopaminergic neuronal death, reported here.

Numerous reasons have been suggested to explain the SNc dopaminergic cell’s susceptibility to Mn. First, the lack of cellular antioxidant defenses, and second, the mitochondrial oxidative energy metabolism disruption [69]. This has led to the assumption that extreme brain Mn levels provoke oxidative stress leading to neurodegeneration [66]. However, the main reason for the specificity of Mn for the SNc dopaminergic neurons is related to the dopamine transporter (DAT); it has been reported that Mn arrives in the neurons via DAT [70, 71, 72, 73]; DAT is related to the MPTP [74], and 6-OHDA neurotoxicity [75], where SNc is more vulnerable than other dopaminergic areas, such as the ventral tegmental area (VTA). It seems that the VTA and SNc dopaminergic cells exhibit topography, biochemistry, and susceptibility to pathological processes differences [76]; the middle and medial SNc express higher levels of DAT than the VTA [74, 77]; thus, Mn possibly get SNc dopaminergic cells through the greater amounts of DAT reported on those neurons. Moreover, in PD, the most vulnerable neurons are the SNc dopaminergic ones and not those of the VTA [76, 77].

Currently, available PD experimental models have contributed significantly to our knowledge of the disease’s neurotransmitter changes, potential neuroprotective therapeutics, and pathophysiology [6, 7]. However, so far, we do not have the most suitable model. MPTP experimental model is the best available one for some reasons, and it has been essential for testing neurorestorative and neuroprotective approaches [4]. Nevertheless, the MPTP model disadvantages are acute damage to the dopaminergic system and infrequent generation of inclusion bodies, different susceptibility among species, and recovery after exposure ceased [78]. Both MPTP and 6-OHDA models differ from the slowly progressive pathology of human PD [4]. In addition, PD genetic models seem to simulate some features of the disease without extensive SNc neuronal loss [79]. Transgenic mice overexpressing wild-type and mutant alfa-synuclein demonstrate motor deficits without loss of dopaminergic neurons [4, 80].

The substantial reduction (72 %) in the number of SNc dopaminergic cells after divalent/trivalent Mn inhalation observed here establishes an evident reduction of this catecholamine content. Therefore, we suppose the motor PD symptoms are due to dopaminergic denervation since L-DOPA-treated animals almost entirely improved their motor performance [27, 28].

6. Conclusion

For some years, PD has shown a significant increase in its prevalence, which is why it has been the focus of multiple investigations worldwide. All these investigations strive to explain the pathophysiological mechanisms of the disease and the best therapeutic alternatives. To accomplish this, we have resorted to the design and use of various animal models, representing an opportunity to study aspects of the disease from different perspectives.

When choosing an animal PD model, the stated objective, scope of the research, and the similarities or discrepancies between the anatomy, physiology, and behavior of humans and animals should be considered. In this chapter, we compared the motor alterations of the model due to unilateral 6-OHDA lesions and inhalation of the MnCl₂/Mn(OAc)₃ mixture.

Our results showed that 6-OHDA unilateral lesion produces acute loss of dopaminergic neurons. These animals did not appear to present bradykinesia and tremor, both clinical aspects that are considered cardinal in PD patients. Unlike 6-OHDA or MPTP PD models, where all symptoms happen in a few days or weeks, or even hours, while in PD patients progress over decades [81]; our Mn mixture-inhaled model seems to be adequate because the symptoms and cell death are bilateral and, and the variances between species are insignificant [25, 26, 27, 28]. According to Schober [78], an ideal PD model must develop the following characteristics: (1) an average number of SNc dopaminergic neurons at birth followed by the gradual and selective loss of these cells beginning in adulthood; (2) easily detectable and quantifiable motor deficits; (3) Lewy bodies; (4) the model, ideally must have a reasonably short time course to simulate the PD pathogenesis (about 3–6 months), which would grant a prompt assessment of therapeutic schemes [78]. Therefore, we replicate at least three of those features with our model [25, 26, 27, 28]. However, additional studies are required to clarify whether Mn-mixture inhalation produces Lewy bodies, reduces striatal dopamine concentrations, and determine if the animals recover after the inhalation.

Finally, our data and the findings of the Mn-model apport crucial knowledge concerning a better understanding of the mechanisms related to the PD nigrostriatal degeneration since it adequately simulates the neurochemical, neuroanatomical, and some behavioral characteristics of PD.

Acknowledgments

This work was supported by COMECyT FICDTEM-2021-066 and PAPIIT-DGAPA IN216821 grants. We are very grateful to Patricia Aley-Medina, Jesús Espinosa-Villanueva and Veronica Rodriguez Mata for their excellent photographic and technical assistance.

Conflict of interest

Authors have declared that no competing interests exist.