Forty years ago, Langston et al. (1983) reported that several young adults in California developed a marked and irreversible parkinsonism shortly after using intravenously a new synthetic illicit drug (Nonnekes et al., 2018). This heroin-like drug resulted to become meperidine, which contained 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a contaminant. The toxicity of MPTP in non-human primates (NHP) displayed clinical signs and nigrostriatal neuropathology similar to the human sporadic PD, henceforth providing an experimental tool for investigating parkinsonism (Burns et al., 1983; Langston et al., 1983).

MPTP as a model of xenobiotic-induced parkinsonism has been studied extensively for many years and constituted the gold standard for experimental PD research. It appeared as a selective neurotoxic xenobiotic inducing dopaminergic nigrostriatal neurodegeneration. As a lipophilic compound, MPTP crosses easily the blood–brain-barrier (BBB) to reach the CNS but it has no toxicity. There it is taken up by astrocytes where MPTP is swiftly oxidized in two steps by monoamine oxidase B (MAO-B) to the neuroactive metabolite 1-methyl-4-phenylpyridinium (MPP+). Pharmacological inhibition of MAO-B protects against MPTP toxicity, while MPP+ systemic administration does not cross the BBB and is only neurotoxic by intracerebral injection. MPP+ released from astrocytes is captured into dopaminergic neurons by the dopamine transporter (DAT) located in the neuronal membrane. This dopamine carrier has a greater expression in neurons of the SN than in other areas and shows a high affinity for MPP+. The final step of MPP+ neurotoxicity consists of the inhibition of complex I activity in mitochondria, resulting in dopamine oxidation to form quinones and reactive oxygen species (ROS) which cause cell death. Due to the high expression of DAT and its high affinity for MPP+ neuronal death is nearly circumscribed to SN and ventral tegmental area, a neuropathology mimicking that of PD (Blesa and Przedborski, 2014).

Different animal species have been and are used to model PD by means of MPTP. Despite its limited use (cost, adequate facilities, technical skills, and ethical issues), NHPs are considered for their close physiology and brain structure with humans the best standard for studies of MPTP-induced parkinsonism (Emborg, 2017; Masilamoni and Smith, 2018; Khan et al., 2023). The NHP model provides the various species of MPTP-treated monkey’s motor, behavioral, and neuropathological features observed in PD patients. However, there are divergences in certain pathophysiological pictures described. The different regimes of treatment may account for the diverse results observed. Thus, NHP treated with acute high doses of MPTP (e.g., 1–4 mg/kg/day, i.p., for 5 days) develop in a short time acute and long-term effects with signs of motor parkinsonism and a clear nigrostriatal loss of dopaminergic neurons and their synaptic terminals in striatum (Waters et al., 1987; Masilamoni and Smith, 2018). In front of this classical treatment, it has been observed that chronic treatment with low doses of MPTP (e.g., 0.2–0.5 mg/kg i.m. once a week) after approximately 21 weeks, and the NHP displayed motor signs non-motor manifestations of PD (Masilamoni et al., 2011). In this chronic study, besides the important nigrostriatal dopaminergic loss, MPTP also caused the loss of non-nigrostriatal dopaminergic neurons and noradrenergic neurons in the locus coeruleus (Oertel et al., 2019), a characteristic pathological feature observed in PD patients. In other experiments, a NHP loss of serotonergic neurons in the dorsal raphe has also been observed (Duperrier et al., 2022).

When comparing the features of MPTP-induced parkinsonism with the neuropathology of patients, the most striking difference is the absence of Lewy bodies and Lewy neurites in the model animals, be NHP or mice.

The most widely used animal model for MPTP-induced parkinsonism is the pigmented C57BL/6 mice. This is the rodent model of choice because rats do not respond to MPTP toxicity and different strains of mice have variable and erratic effects to its exposure (Tello et al., 2022; Khan et al., 2023). The neurotoxic action of MPTP on C57BL/6 mice follows the same pattern as described above for NHP. Here again, the rodent model shows certain variability according to the dose level and the time span of treatment (Merghani et al., 2021; Khan et al., 2023). In this way, mice MPTP-treated with 30 mg/kg/day i.p. for 6 days (a dose much greater than those used with NHP) developed degeneration of the nigrostriatal pathway in both the SN and the striatum, as shown by loss of dopaminergic neurons immunostained for tyrosine hydroxylase (TH), depletion of striatal dopamine, activation of glial cells in the nigrostriatal pathway, and impairment in behavioral tests (Bhurtel et al., 2019). It has been proposed that oxidation of dopamine plays a role in the loss of dopaminergic neurons in SN. However, pharmacological depletion of dopamine does not protect against nigrostriatal degeneration in MPTP-treated C57BL/6 mice (Hasbani et al., 2005).

In humans, sex differences have been reported for PD, especially a higher incidence in men than in women (Miller and Cronin-Golomb, 2010). The MPTP model in NHP has been carried out in female (Waters et al., 1987; Masilamoni et al., 2011) and male (Burns et al., 1983; Masilamoni and Smith, 2018), but to the best of our knowledge, no sex differences were studied. Most research using C57BL/6 mice includes only male subjects; nevertheless, comparison of male and female mice treated with small, chronic doses of MPTP revealed selective motor impairment only in male subjects (Antzoulatos et al., 2010).

Rotenone is a toxic isoflavone present in certain plants of the genera Derris and Lonchocarpus. It has been used as a broad-spectrum ichthyotoxin, insecticide, and pesticide. The epidemiological observation that chronic exposure to systemic pesticides such as rotenone is associated with an increased risk of PD led to investigate whether rotenone produces in experimental animal manifestations of PD (Betarbet et al., 2000). The positive results of that research established the rotenone model, since then, one of the most employed in PD studies.

Rotenone as a lipophilic compound crosses the biological membranes, including the BBB and, in contrast with MPTP, acts directly in the cell inhibiting the electron transport chain of the mitochondrial complex I (NADH: ubiquinone oxidoreductase). This effect reduces ATP formation and can produce ROS and lead to cell death. In contrast with MPTP, there is no selective toxicity. Rotenone acts as a toxin on all cells of the organism, and the CNS manifestations are related to the greater vulnerability of certain systems. The chronic inhibition of mitochondrial complex I by rotenone determines a highly selective nigrostriatal dopaminergic degeneration associated with motor signs (hypokinesia and rigidity). A specific mark of the rotenone-treated rats is that nigral neurons accumulate fibrillary cytoplasmic inclusions containing α-synuclein and ubiquitin, morphologically similar to Lewy bodies (Johnson and Bobrovskaya, 2015).

The original rotenone model, briefly seen above, established an optimal dose for producing the PD pathology: 2–3 mg/kg/day administered by osmotic pump in Lewis rats for periods of 7 days to 5 weeks of continuous administration. Motor and behavioral tests, biochemistry, and neuropathology defined the model. Nonetheless, a great number of variants in animal species, dosage, via and span of administration, and methods of evaluation have appeared over time. Numerous articles testing drugs or biologicals use a quick behavioral or motor test in a short-term, high-dose pseudorotenone model. It is well known that high doses of rotenone, up to 100 mg/kg, for a short period produced systemic toxicity and unspecific lesions in the brain such as necrosis in the striatum. The ability of the different rotenone model variants to reproduce the clinical features of PD has been analyzed in various reports (Johnson and Bobrovskaya, 2015; Tello et al., 2022; Khan et al., 2023).

A question concerning the usefulness of the MPTP and rotenone models in inducing the clinical neurological signs and neuropathology of sporadic PD is still open. Two recent studies have addressed the issue by comparing experimentally both models. Bhurtel et al. (2019) treated groups of four C57BL/6 mice with MPTP 30 mg/kg/day i.p. or with rotenone 2.5 mg/kg/day i.p. for 6 days and compared the neurotoxicity of both compounds. A battery of behavioral tests, neurochemical analysis, and immunohistochemical staining were applied to both treated groups. It was determined that MPTP induces dopaminergic nigrostriatal lesions with neuronal loss and behavioral impairment as it has been seen above, but not rotenone (at least at the dosing level administered). The rotenone group only displayed neurodegeneration and glial activation in the hippocampus. The authors propose that MPTP is more specific than rotenone to produce dopaminergic neurodegeneration.

In contrast, in a comprehensive study, Zhang et al. (2022) dosed groups of 30 C57BL/6 mice with MPTP 20 mg/kg i.p. twice a week over 6 consecutive weeks, or with rotenone 30 mg/kg by gavage following the same schedule. To compare the effects of both neurotoxic agents, neurobehavior, neuropathology, neurochemistry, and mitochondrial function were assessed in treated and control groups by means of a large and complete battery of adequate techniques. Locomotor activity and coordination and exploratory behavior were significantly lower in both treated groups compared with controls. The MPTP-treated group showed the typical loss of dopaminergic neurons in SN pars compacta and the reduction of TH content in SN and striatum that were more significant than in the rotenone group. Remarkably, oxygen consumption in mitochondrial complex I in the SN was significantly reduced in the rotenone group compared with the MPTP group. As expected, Lewy bodies were only present in SN neurons in the rotenone group.

The striking difference in the rotenone effects observed in these studies may belong to the low dose of rotenone, via administration used, and duration of treatment. After pondering the different procedures described in the literature for the rotenone model, it appears as more reproducible and consistent those described by Betarbet et al. (2000) and Zhang et al. (2022).

The rotenone model has also provided an experimental approach to the hypothesis considering sporadic PD as a staging disease that can start in the ENS and progress through the nervous system until reaching the brain (Braak and Del Tredici, 2008; Braak and Del Tredici, 2017). In relation to that concept, the administration of rotenone intragastrically to C57BL/6J mice 5 mg/kg 5 days a week for 1.5–3 months induced α-synuclein accumulation in all the nervous system structures considered for the progression of PD. Namely, it starts in the ENS and follows the dorsal motor nucleus of the vagus, the intermediolateral nucleus of the spinal cord and the SN. α-synuclein phosphorylation in the ENS and other nervous structures was also observed. The alterations are sequential appearing only in synaptically connected structures. The rotenone model, then, may be apt for studying the α-synucleinopathies (Pan-Montojo et al., 2010).

The rotenone model has been commonly studied in male rats and mice. Related to sex, one study reported that rotenone-treated male rats show a decrease in testosterone levels in blood plasma (Alam and Schmidt, 2004). This decrease is one of the comorbidity signs found in male PD patients (Okun et al., 2002) and is probably linked to the more severe motor deficits in male rodents (Antzoulatos et al., 2010). Furthermore, sex differences have been shown in female rats that display a decreased sensitivity to rotenone-induced neurodegeneration (De Miranda et al., 2019). These female subjects showed less inflammation and less accumulation of α-synuclein than matched males. Sex differences in sensitivity may reflect the higher male ratio in human PD.

6-Hydroxydopamine (6-OHDA) is a neurotoxic xenobiotic analogue to the catecholaminergic transmitters. Its systemic administration produces a long-lasting depletion of noradrenaline and dopamine in peripheral nerve terminals but not in the CNS as it does not traverse the BBB. Injecting intracerebrally 6-OHDA in experimental animals is the oldest method of modelling PD (Ungerstedt, 1968). Although with many refinements added over time, 6-OHDA continues to be used to investigate the pathophysiology of PD as well as the preclinical aspects of putative neuroprotective or therapeutic drugs (Simola et al., 2007; Hernandez-Baltazar et al., 2017). By means of stereotaxic surgery, 6-OHDA is injected into the medial forebrain bundle that destroys the dopamine neurons projecting from the midbrain to the striatum. To increase the selectivity of 6-OHDA for dopaminergic neurons, animals must be treated previously with a blocker of the noradrenaline transporter, usually with desipramine. Stereotaxic administration of 6-OHDA is also done in the striatum, causing a more progressive and less severe lesion. Most studies on the motor deficits of PD use the ipsilateral injection of 6-OHDA in the right medial forebrain bundle or in the right mid-striatum to obtain the model of contralateral rotational behavior. This model works satisfactorily in many species (monkeys, rats, and mice). An application of interest is the use of the mouse model of rotation for tissue transplantation in the damaged zone, an area of stem cell research (Bagga et al., 2015; Guimarães et al., 2021). The 6-OHDA model in its rotational version is also employed in transgenic mice to search for very specific aspects of dyskinesias (Castela et al., 2023).

The mechanisms of neurotoxicity and usefulness of the 6-OHDA model are to a great extent similar to those of MPTP although in several aspects 6-OHDA can provide more precise and detailed neuropathological information. Both models do not express α-synuclein, but in a recent study (Cui et al., 2023), 6-OHDA injected in the rat medial forebrain bundle did not induce expression of α-synuclein in the brain, but at 5 weeks, post-lesion were detected as having increased levels of phosphorylated α-synuclein in ileum and colon. That finding may be related to the multistage theory seen above on the axis gut–brain for sporadic PD.

Male rats and mice are the customary animals used in the 6-OHDA model. A study of bilateral partial lesions with 6-OHDA in rats of both sexes observed a reduction of locomotor activity in aged male subjects but not in female subjects (Cass et al., 2005). Moreover, it has been shown that the effects of 6-OHDA striatal lesions on spatial cognition are sex-specific in rats and testosterone-dependent in male subjects (Betancourt et al., 2017).

Scopolamine is a tropane alkaloid found in several plants. It is a cholinergic neurotoxin due to its properties as a competitive antagonist of the muscarinic receptors. At the right dose, it is used as anti-emetic drug. Scopolamine is lipophilic and easily crosses the BBB causing a powerful effect in the CNS. Single or repeated i.p. administration of scopolamine in mice and rats induces cholinergic dysfunction and increase of AChE that further cleaves acetylcholine at the synapses leading to amnesia and cognitive loss. Doses administered range from 0.4 mg/kg to 3 mg/kg i.p., with longer-lasting memory impairment after repeated daily administration for one or more weeks than a single injection (Rahimzadegan and Soodi, 2018; Chen and Yeong, 2020; Kose et al., 2023). This model is mainly used for behavioral studies, but some authors report that scopolamine induces an imbalance of the amyloid-beta precursor protein (APP) processing to the amyloidogenic pathway causing intraneuronal accumulation of amyloid-beta (Aß) and also increased tau synthesis (Chen and Yeong, 2020; Kose et al., 2023). Noticeably, the scopolamine model in mice and, to a minor extent, in rats, was found the most widely used in vivo model for multitarget anti-AD drug discovery in a bibliometric analysis of the last 3 decades (Sampietro et al., 2022). Male and female rats experience a similar increase in AChE and memory loss in response to scopolamine (Ali and Arafa, 2011) although sex differences in fine cholinergic responses cannot be discarded.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is widely used to induce neuroinflammation in rats and mice. There are a variety of protocols using different LPS serotypes and doses ranging from 0.3 mg/kg to 10 mg/kg i.p. and 0.25 μg to 50 μg i.c.v., with single or repeated administration over one or more days (Nava Catorce and Gevorkian, 2016; Zakaria et al., 2017). The systemic treatment through i.p. injection in mice is very convenient for testing protective anti-inflammatory agents (Nava Catorce and Gevorkian, 2016). The sickness behavior response of mice to LPS peripheral infection parallels the release of proinflammatory cytokines in the brain and blood (Molina-Martínez et al., 2021). The mechanisms of microglia activation by LPS have been fully characterized either after peripheral (i.p.) or central (i.c.v.) administration in mice and rats. LPS elicits a robust neuroinflammatory response followed by cognitive loss and neurodegeneration although the response and the brain areas affected may differ according to the administration regimen and the age of the animal (Nazem et al., 2015; Zakaria et al., 2017; Decandia et al., 2023). Furthermore, some clinical evidence supports an involvement of LPS from Gram-negative bacteria resident in the gastrointestinal tract as a potential contributor to the onset of AD (Zhao et al., 2017). Rodents dosed peripherally with LPS make a reliable model of AD neuroinflammation. In addition, LPS may promote increased brain Aβ levels and tau pathology although the wide diversity in the treatment design and the LPS serotypes used do not yield clear conclusions (Batista et al., 2019). Male and female rodents are used with good results (Nava Catorce and Gevorkian, 2016). However, there are sex differences in the pattern of immune responses to LPS treatment that should be taken into account when evaluating proinflammatory biomarkers (Sharma et al., 2018).

Streptozotocin (STZ) is a naturally occurring glucosamine-nitrosourea compound identified in a Streptomyces bacteria strain. The first use of this toxin is to model diabetes in rodents as peripheral injection of STZ damages pancreatic β-cells. Impaired insulin sensitivity is indeed a risk factor for AD development. In the brain, both i.p. and i.c.v. injections of STZ cause neuroinflammation and cognitive deficits in mice and rats (Nazem et al., 2015). Interestingly, bilateral i.c.v. injections of STZ in mice elicit chronic neuroinflammation and increased levels of Aβ and hyperphosphorylated tau (p-tau) in the hippocampus (Fan et al., 2022). Therefore, centrally administered STZ, at doses 1 or 3 mg/Kg i.c.v., is considered a better model of sporadic AD than peripherally administered STZ (Nazem et al., 2015; Fan et al., 2022). A sex-difference study reported that female rats respond less than male subjects to i.c.v. STZ (Bao et al., 2017), and thus, female subjects might not be suitable as the STZ model of AD.

Monomeric C-reactive protein (mCRP) is a proinflammatory molecule yielded by activation and disaggregation of the pentameric CRP. Clinically, it has been associated with the triggering and progression of AD through its deposition in stroke-damaged areas (Slevin et al., 2015, 2020). A recently developed mouse model of dementia by intrahippocampal injection of mCRP may be a new tool to study AD and neuroprotective agents (García-Lara et al., 2021). C57BL/6 J mice injected bilaterally with a dose of 3.5 μg of mCRP show a long-lasting loss of learning and memory. Remarkably, cognitive loss was protected by the oral administration of an anti-inflammatory drug (García-Lara et al., 2021). The mCRP dementia model shows a marked increase in p-tau levels in the hippocampus area (Slevin et al., 2015; García-Lara et al., 2021), whereas a slight increase in Aβ was detected by immunostaining (Slevin et al., 2015). Sex differences have not yet been analyzed in this new model.

The rodent models generated with the marine toxin okadaic acid and the neurotoxic metal aluminum are less characterized regarding neuroinflammation. Okadaic acid inhibits selective protein phosphatases. It would induce neuroinflammation and associated memory impairment after i.c.v. injection, but it is mainly used as a model of tau hyperphosphorylation in rats and mice (Nazem et al., 2015). No clear sex differences in susceptibility to okadaic acid have been reported.

Furthermore, administration of aluminum chloride in rats induces brain damage and cognitive impairment, but the underlying mechanisms are not clarified and may involve activation of tau phosphorylation enzymes (Sanajou et al., 2023). Despite suggestions of gender bias in the effect of aluminum in humans, no sex differences have been reported in rodent models.

Finally, neuroinflammation induced by a high-fat diet (HFD) in rodents is also a model of AD neurodegeneration, where neuroinflammation and oxidative stress trigger tau and amyloid pathology and cause cognitive impairment (Sharma, 2021). Several studies have analyzed the effects of antiinflammatory treatments against HFD (Zameer et al., 2020; Sarroca et al., 2021). HFD induces higher neuroinflammation and metabolic impairment in male subjects than female subjects, and this dimorphism likely causes a sex bias as a model of AD (see Sharma, 2021).

Neuroinflammation plays a role in PD although it may be less decisive for the neurodegeneration triggering and development than in AD. Studies in animal models suggest that neuroinflammation exacerbates the outcome of neurotoxicity induced by specific dopaminergic toxins (Ramsey and Tansey, 2014). Animal models based on the combination of central and peripheral inflammatory challenges show microglial activation and death of dopaminergic neurons after treatment with proinflammatory agents, such as LPS, paired with neurotoxic agents as paraquat and rotenone (García-Revilla et al., 2022).