Degeneration of dopaminergic neurons of the nigrostriatal pathway leads to Parkinsonian motor deficits

The mechanistic understanding of striatal DA and its regulatory role in the extrapyramidal motor control system is well established (Alexander et al. 1986; Penney et al. 1986; Albin et al. 1989; DeLong et al. 1990; Obeso et al. 2008; Blandini et al. 2000). The selective degeneration of DA neurons in the SNpc (and the subsequent decline in striatal DA levels) have been known to be linked to PD symptoms for more than 50 years (Ehringer et al. 1960). The reduction of DA in the striatum is characteristic for all etiologies of PD (idiopathic, familial, chronic manganese exposure) and related parkinsonian disorders (Bernheimer et al. 1973), and it is not observed in other neurodegenerative diseases, such as Alzheimer’s or Huntington’s Diseases (Reynolds et al. 1986). In more progressive stages of PD, not only a loss of DA neuronal terminals in the striatum, but also a degeneration of the entire DA neuron cell bodies in the substantia nigra pars compacta (SNpc) was detected (Leenders et al. 1986; Bernheimer et al. 1973). The different forms of PD exhibit variations in the degradation pattern of the SNpc DA neurons. In idiopathic PD, for example, the putamen is more severely affected than the caudate nucleus (Moratalla et al. 1992; Snow et al. 2000). All different PD forms however are characterized by the loss in striatal DA that is paralleled by impaired motor output (Bernheimer et al. 1973). Characteristic clinical symptoms of motor deficit (bradykinesia, tremor, or rigidity) of PD are observed when more than 80 % of striatal DA is depleted (Koller et al. 1992). These findings on the correlation of a decline in striatal DA levels as a consequence of SNpc DA neuronal degeneration with the onset of clinical PD symptoms in man provide the rationale for the current standard therapies that aim to supplement striatal DA, either by the application of L-DOPA, or by a pharmacological inhibition of the endogenous DA degradation-enzyme monoaminde oxidase B (MAO-B). These treatments result in an elevation of striatal DA that is correlated with an improvement of motor performance (Calne et al 1970). The success of these therapies in man as well as in experimental animal models clearly confirms the causal role of dopamine depletion for PD motor symptoms.

The experimental support linking the degeneration of DA neurons of nigrostriatal pathways with the manifestation of motor symptoms characteristics of parkinsonian disorders comes from human clinical observations as well as from primates, mice and rat in vivo models using DA neuron ablation by toxicants. The levels of striatal DA corrected with the onset of PD symptoms, and dopaminergic degeneration preceed the onset of motor symptoms. The exemplary animal studies selected here are based on the use of MPTP or rotenone. The efficacy of MPTP or rotenone treatment depends on the regimen applied (acute, subacute, chronic administration), the age of the animals, and the strains used. For the interpretation of the studies, it is important that in some animal models the initial depletion of DA is only partially explained by neurite degeneration. The other contributing factors are downregulation of TH, and depletion of DA from synaptic terminals. These effects recover after 1-2 weeks. This makes the time point of measurement important for the correlation of effects. Moreover, the mouse brain has a very high plasticity after damage, so that motor deficits can recover after several weeks although there is pronounced dopaminergic neuro degeneration.

Rat in vivo models

• Rat/rotenone: Correlation between striatal DA, SNpc DA neurons, and motor deficits. Lewis rats exposed to systemic rotenone (3 mg/kg/ day i.p.) exhibited a loss of TH positive neurons in the SNpc by 45 %. Motor deficits were assessed by the postural instability test and by the rearing test. While 3 month old animals developed motor symptoms after 12 days of rotenone exposure, 7 month and 12 month old animals developed motor symptoms already after 6 days of exposure. Rotenone treatment elicited a progressive development of motor deficits that was reversible when treated with a DA agonist. Similar to that, the loss of rearing performance evoked by rotenone was reversed by the DA agonist apomorphine. Rotenone elicited terminal loss in the dorsolateral structures. While in the dorsolateral striatum, a significant loss of TH-positive neurites was detected, striatal cell bodies were spared from degeneration. Initial striatal DA levels (75 ng/ mg protein) dropped to 45 % following rotenone treatment (Cannon et al. 2009).

• Rat/6-OHDA: Destruction of nigrostriatal DA neurons. Unilateral injection of 6-OHDA into the dopaminergic nigrostriatal pathway leads to a preferential loss of DA neurons that is correlated with the onset of rotational motor deficits (Luthman et al. 1989; Perese et al. 1989; Przedborski et al. 1995).

• Rats/rotenone: Correlation between striatal dopamine and motor symptoms; partial reversibility by L-DOPA. Rats were exposed to 2.5 mg/kg rotenone, daily, for 48 days. Dopamine detected in the anterior striatum and posterior striatum was reduced by ca. 50 % after rotenone treatment. Rotenone treatment resulted in a significantly prolonged descent latency compared to control in the bar test and grid test. In the catalepsy test, descent latency dropped from 35 s of the controls to 5 s. In the grid test, a reduction from 30 s (control) down to 4 s (rotenone) was observed. The average distance travelled within 10 min by the animals was reduced from 37 m to 17 m in the rotenone group. Average number of rearings declined from 65 to 30; the time of inactive sitting of 270 s in controls was increased to 400 s in the rotenone group (Alam et al. 2004).

• Rat/rotenone: Correlation between striatal dopamine and motor symptoms. Rats were treated with rotenone either at doses of 1.5 mg/kg or 2.5 mg/kg over two months with daily i.p. injections. In the 2.5 mg/kg group, striatal DA levels dropped from 6400 pg/mg in the controls to 3500 pg/mg in the rotenone group. Rotenone treated animals showed an extended descent latency (5 to 50). In a vertical grid test, latency time increased from 9 s to 72 s (Alam et al. 2002).

• Rats/rotenone: Correlation between nigrostriatal TH intensity and motor symptoms. Rats were treated with different doses of rotenone for 21 days with daily i.v. or s.c. injections. In the 2.5 mg/kg group, TH intensity in the striatum dropped from 0.2 to 0.12. The average time to initiate a step increased from 5 s in the controls to 11 s in the rotenone group. Spontaneous rearing scores dropped from 80 % of the vehicle treated controls to 20 % in the rotenone group (Fleming et al. 2004).

• Rat/rotenone: In middle-aged rats exposed to rotenone (3 mg/kg/day for 6 days), a reduction of striatal DA levels and TH positive neurons by ca. 50 % correlated with impairments rearing performance and postural instability tests (Cannon et al. 2009).

• Rat/rotenone: In rats, exposed to rotenone (2.5 mg/kg/day), spontaneous locomotor activity was reduced by ca. 50 % after 1 week of rotenone treatment. This impaired motor performance was correlated with a loss of striatal DA fibers by 54 % and a loss of nigral DA neurons by 28.5 % (Höglinger et al. 2003).

Mouse in vivo models

• Mouse/MPTP: In mice exposed to MPTP in combination with probenecid, both a chronic treatment scheme (MPTP 25 mg/kg, in 3.5 day intervals for 5 weeks) as well as a subacute treatment scheme (25 mg/kg, 1x per day for 5 days) resulted in a deletion of striatal DA that was directly correlated with impairments in motor symptoms (Petroske et al. 2001).

• Mouse/MPTP: In a mouse model exposed to MPTP at 15 day intervals (36 mg/kg), lower rotarod performance was observed after the fourth injection. The decline in motor performance was correlated with the decline in TH-immunoreactivity in the striatum (r2 = 0.87) (Rozas et al. 1998).

• Mouse/D2 receptor knockout. Mice deficient in D2 receptors displayed akinesia, bradykinesia and a reduction in spontaneous movement (Baik et al. 1995).

Monkey in vivo models

• Monkey/MPTP: Correlation between striatal DA, SNpc DA neuron number and PD symptoms. Macaca exposed to MPTP (i.v) (0.2 mg/kg, daily) display signs of PD at day 15, including motor abnormalities. The transition between the presymptomatic and symptomatic period occurred between day 12 and day 15 of MPTP exposure. At day 15, TH neurons in the SNpc were reduced by 50%, DAT binding autoradiography studies revealed a decline in binding also by 50% at day 15. Compared with control values of 150 pg/µg protein, the DA content of the caudate nucleus dropped to values < 10 pg/µg protein at day 15. In the putamen, DA levels dropped from 175 pg/µg protein to 20 pg/µg protein at day 15 (Bezard et al. 2001).

• Monkey/MPTP: Correlation between striatal DA, SNpc DA neurons, and PD symptoms. Monkeys display a motor symptom pattern similar to that observed in humans. In order to optimize a MPTP intoxication protocol that allows a gradual development of nigral lesion, different states of PD symptom severity were defined and correlated with the amount of striatal DA and the number of TH-positive neurons in the SNpc. Asymptomatic monkeys displayed a reduction in striatal DA by 30 %, a neuronal loss in the SNpc by 40 %, and a decline in striatal expression of TH, DAT and VMAT2 by 50-60 %. Monkeys that recovered from early PD symptoms displayed a reduction of striatal DA of 50 %, a loss of TH neurons in the SNpc and a loss of DAT and VMAT2 expression up to 60 %. In animals with moderate PD symptoms, striatal DA levels as well as TH positive neurons and DAT and VMAT 2 expression were reduced by 70-80 %. Animals with severe PD symptoms displayed remaining levels of striatal DA and SNpc expression of TH, DAT and VMAT2 of around 20 % compared to untreated controls (Blesa et al. 2012).

• Monkey/MPTP: The established model of basal ganglia wiring received ample experimental support in recent years. For instance, an increase in the inhibitory output by GPi/STN has been observed in MPTP treated monkeys, similar to the situation in idiopathic PD patients. These findings were corroborated by observations indicating an elevated mitochondrial activity and an elevated firing rate of the inhibitory output nuclei detected on the level of individual neurons (Mitchell et al. 1989; Filion et al. 1991). Lesions in the output ganglia of monkeys lead to a reduction in the output and to an improvement in motor control (Bergman et al. 1990; Aziz et al. 1991). In analogy to these lesion experiments, deep brain stimulation of these regions results in a profound improvement of motor performance in PD patients (Limousin et al. 1999; Ceballos-Baumann et al. 1994).

Human PD

• Human PD: Association of PD phenotype with impaired striatal DA. In the brains of human PD patients, a significant decrease of striatal DA was observed (Lloyd et al. 1975). In the caudate nucleus, levels of DA dropped from control values of 4 µg/g tissue to levels of 0.2 µg/g. In the putamen, control values were in the range of 5 µg/g and 0.14 µg/g in the PD patient group. The levels of DA in the striata of DA patients that received L-DOPA treatment was 9-15 times higher compared with non-treated PD cases.

• Human PD: Correlation between striatal DA loss and degeneration of DA neurons in the SNpc. Examinations of the brains of PD patients revealed morphological damage in the SNpc, accompanied by the degeneration of DA neurons (Earle et al. 1968).

• Human: Association of striatal DA levels and motor performance. In order to substitute degenerated DA neurons in the SNpc, human fetal tissue from the ventral mesencephalon was transplanted to the caudate and putamen in idiopathic cases PD as well as in patients that developed PD-related motor deficits as a consequence to MPTP intoxication. Transplanted cells led to a reinnervation of the striatum with DA projections (Widner et al. 1992; Kordower et al. 1995, 1998). In these case studies, patients demonstrated a sustained improvement in motor function (decline in rigidity score by more than 80 %).

• Human PD: correlation between nigrostriatal DA neuron content and motor symptoms. Imaging of DAT was performed by the use of 123I-FP-CIT SPECT (single photon emission computed tomography). Clinical PD severity was determined by using the Unified Parkinsons Disease Rating Score (UPDRS). In PD patients, DAT binding in the striatum, caudate, and putamen correlated with disease severity and duration of disease (Benamer et al. 2000).

• Human PD: correlation between 18F-dopa uptake measured by PET and the onset of motor symptoms detected according the UPDRS. 18F-dopa influx rate constants (Ki/min) were reduced in the midbrain from 0.008 to 0.006, in the right putamen from 0.017 to 0.0036, and in the left putamen from 0.017 to 0.005 (Rakshi et al. 1999).

• Human PD: correlation between putamen influx rate (Ki/min). Ki (control): 0.0123; asymptomatic PD (no observable motor deficits): 0.0099; symptomatic PD (clinically evident motor deficits): 0.007. Mean UPDRS value was "15.1  7.5". A correlation coefficient of -0.41 was detected between motor UPDRS and putamen influx (Ki) (Morrish et al. 1995).

• Human PD: Correlation of the degree of monoaminergic degeneration in early PD with motor symptoms assed by the UPDRS and the Hoehn and Yahr Stage scale. For PET imaging, 18F-9-fluoropropyl-dihydrotetrabenzazine that targets VMAT2 was used. Uptake of the tracer was reduced by 20-36 % in the caudate, by 45-80 % in the putamen, and by 31 % in the substantia nigra. This correlated with a total UPDRS value of "12.1  7.1" in the PD group, respectively with a HY value of "1.0  0.1" in the PD group compared to controls (Lin et al. 2014).

• Human PD: Correlation between the decline in 18F-dopa rate constant (Ki) and the onset of motor deficits. The 18F-dopa rate constant Ki was reduced in the caudate nucleus (0.011 down to 0.0043) and inversely correlated with an increase in the UPDRS from "11.9  5.2" to "50 11.6" (Broussolle et al. 1999).

• Human PD: Correlation between striatal DAT binding measured by the use of 123I-CIT SPECT and motor deficits. A correlation coefficient between 123I-CIT binding and UPDRS motor scale of -0.56 was detected. A correlation coefficient of -0.64 between 123I-CIT binding and Hoehn and Yahr stage scale was detected. Motor symptoms in the clinically less affected body side show a closer correlation with striatal DAT binding (Pirker et al. 2003).

• Human PD: Correlation between the reduction in the putamen uptake of 18F-CFT and the severity of PD motor symptoms. 18F-CFT uptale was reduced to 18 % in the putamen, to 28% in the anterior putamen, and to 51 % in the caudate nucleus (Rinne et al. 1999).

• Human PD: Reduction in 123I-CIT binding in the putamen by 65 % correlated with a mean UPDRS score of 27.1 (Tissingh et al. 1998).

• Association between striatal DA and motor performance. Application of L-DOPA leads to a substitution of DA in the striatum and improves motor performance. (Boraud et al. 1998; Gilmour et al. 2011; Heimer et al. 2002; Papa et al. 1999; Hutchonson et al. 1997; Levy et al. 2001).

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Deguelin

In experimental rat models, chronic subcutaneous infusion of deguelin at 6 mg/kg/day for up to 14 days induced selective degeneration of the nigrostriatal dopaminergic pathway (Caboni et al. 2004). This was evidenced by reduced tyrosine hydroxylase (TH) immunoreactivity in the striatum and substantia nigra, along with silver staining indicative of degenerating nerve terminals. Affected animals exhibited hypokinesia, unsteady gait, hunched posture, and rigidity in some cases, mimicking PD-like motor symptoms. At a lower dose (3 mg/kg/day), deguelin did not produce significant neurodegeneration, highlighting a dose-dependent effect. Overall, about 50% of rats treated at the higher dose developed PD-like lesions. Biochemical analysis revealed that the the neuropathology rating observed after exposure to 6 mg/kg b.w. (14 days) correlate with the brain concentration of the parent compound (0.24–0.39 ppm), not its metabolites (Caboni et al. 2004). -

Paraquat

• Paraquat treatment (10 mg/kg twice a week for 4 weeks) of young adult Sprague-Dawley rats (2 months old) induced a significant loss of nigral dopaminergic neurons (by Nissl staining and TH immunostaining) in SNpc of 15% and a mixed pattern of motor impairments (postura deficit, decrease in speed and mobility), which may have been related to early effects of nigral dopaminergic neuronal loss (Cicchetti et al., 2005).
• Adult C57 BL/6 mice treated i.p. with paraquat (5 and 10 mg/kg) showed a dose-dependent decrease in substantia nigra dopaminergic neurons (36% and 61%, respectively, assessed by Fluoro-gold prelabelling method), a decline in striatal dopamine nerve terminal density (87% and 94%, respectively, assessed by TH immunoreactivity) and neurobehavioural syndrome characterised by reduced ambulatory (locomotor) activity (Brooks et al., 1999).
• Paraquat treatment (i.p. 10 mg/kg twice a week for 4 weeks) of male Swiss Albino mice, 22–14 weeks old, induced progressive motor dysfunction with severe postural instability and gait impairment. A concomitant decrease in the expression levels of TH in SN (approximately 60%), FC (frontal cortex) and hippocampus and a decrease (approx. 40%) in TH+ and FOX3+ neurons in SN were observed (stereological evaluation). As part of the toxicological evaluation of the most suitable sublethal dose, mice were also treated at 5 mg/kg by i.p. twice a week for 4 weeks. In addition, a decrease in DOPA-decarboxylase was observed in the SN and FC. The only endpoint measured (in addition to the general toxicity endpoints) was the neuronal countin the SN. A statistical signi!cant decrease (approximately 15%) in TH+ and FOX3+ neurons was observed (Mitra et al., 2011).
• Male C57BL/6 mice, 6 weeks, 5 months and 18 months old, were i.p. treated with paraquat at 10 mg/kg twice a week for 3 weeks (6 injections in total). Age-dependent reduction in locomotor activity and motor coordination was observed. The 18-month old mice were themost severely affected and failed to recover 24 h post treatment. Progressive reduction indopamine metabolites and turnover were greatest in the 18-month old group of animals.Increased in striatal TH activity was observed in the 6-week-old and 5-month-old animals butnot in 18-month-old mice. The number of nigrostriatal dopaminergic neurons was reduced inall age group animals but these losses, along with the decreases in striatal TH protein levels,were progressive in 18-month-old paraquat groups between 2 weeks and 3 months postexposure.(Thiruchelvam et al., 2003).
• Intracerebral injection of 1–5 lg paraquat in male Wistar rats (3 months old) for 16 weeks caused dose-dependent depletion DOPA in the ipsilateral striatum starting 2 weeks after treatment (long-lasting and irreversible) up to 91.5% at 3 lg paraquat. Paraquat inducedmarked loss of Nissl substances and severe loss of neurons at 3 lg. PQ caused dosedependent rotational behaviour in rats, contralateral to the lesion side, in response toapomorphine administration (inducing circling behaviour) (Liou et al., 1996).
• Male Wistar rats were injected with 10 mg/kg paraquat i.p. for 4–24 weeks. Paraquat induced reduction in TH+ neurons of the SN (17% at 4-week mainly in the rostral region, up to 37% at 24 weeks expanding to the whole length of SN; evaluated by stereology). DOPA levels increased in the caudate-putamen (4–8 weeks) then returned to control values and dropped (25–30%) after 24 weeks. This seems to result from degeneration of DA neurons. TH level (Western blot) decreased in the caudate-putament after 24 weeks (55%) but this effect was not rejected by the loss in TH-ir neurons (being already dropped in the rostral part of SN after 4 weeks) (Ossowska et al., 2005). Clinical signs were not recorded in this study; however the study design was considered of relevance for the evaluation of the progression of the finding associated with neuronal loss.
• Paraquat treatment (i.p. injection 10 mg/kg bw every 5 days over 20 days) of Long Evans Hooded rats induced progressive (TH positive neurons stereology counted) loss in dopaminergic neurons up to 47% (end of week 8 post PQ exposure) and de!ciency in behavioural motor function (horizontal beam walking test) (after 4 and 8 weeks). Ubisol-Q10 (6 mg/bw) administration after completion of paraquat injections (when the degenerative process had already began (20% TH positive neurons lost)) was effective in blocking the progression of neurodegeneration and improved motor skills. To maintain this neuroprotection, continuous Ubisol-Q10 supplementation was required. Discontinuation of treatment resulted in neuronal death, suggesting that the presence of the antioxidant was essential for blocking the pathway (Muthukumaran et al., 2014).
• In Fernagut (2007) experiment, male mice overexpressing human a-syn under the Thy 1 promoter (Thy 1-aSYN) and WT were i.p. injected PQ 10 mg/kg once a week for 3 weeks. Despite degeneration of dopaminergic neurons (densitometric measurement and stereological analysis for counting TH+ neurons) in both Thy 1-aSYN mice and WT PQ-treated mice, behavioural impaired sensimotor performance was observed in non-treated Thy 1-aSYN mice only, remaining unchanged after PQ administration. The sensimotor abnormalities in Thy 1-aSYN were observed in a previous work (Fleming et al., 2004) and the lack of behavioural deficits after PQ administration was commented by the author as not surprising in the view of small magnitude neuronal loss TH-positive terminals in striatum (25%).

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• Motor abnormalities observed in PD display large interindividual variations.

• The model of striatal DA loss and its influence on motor output ganglia does not allow to explain specific motor abnormalities observed in PD (e.g. resting tremor vs bradykinesia) (Obeso et al. 2000). Other neurotransmitters (Ach) may play additional roles.
• There are some reports indicating that in subacute rotenone or MPTP models (non-human primates), a significant, sometimes complete, recovery of motor deficits can be observed after termination of toxicant treatment. While the transient loss of striatal DA can be explained by an excessive release of DA under acute toxicant treatment, the reported losses of TH-positive neurons in the SNpc and their corresponding nerve terminals in the striatum are currently not explained (Petroske et al. 2001).
• In MPTP treated baboons, the ventral region of the pars compacta was observed to be more severely degenerated that the dorsal region. This pattern is similar to the degeneration pattern in idiopathic PD in humans. These observations indicate that two subpopulations of nigrostriatal DA neurons with different vulnerabilities might exist (Varastet et al. 1994).

• According to the classical model of basal ganglia organization, DA is assumed to have a dichotomous effect on neurons belonging either to the direct or indirect pathway. More recent evidence however rather indicates that D1 and D2 receptors are expressed on most striatal neurons in parallel (Aizman et al. 2000).
• Large variability exists regarding the onset of the downstream AO. This is dependent upon the the stressor used and the route of exposure and variability in the experimentl outcome consequent to differences in the route of exposure is a frequent inconsistencies.

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Uncertainties or inconsistencies

• Exposure to paraquat may decrease the number of nigral neurons without triggering motor impairment (Fernagut, 2007). This can be consequent to the low level of DA reduction or limited neuronal loss observed following the treatment.
• The impact of paraquat upon the striatum appears to be somewhat less pronounced than the effects of the pesticide upon SNc DA neuronal soma (Mangano et al., 2012). As well, some authors have failed to !nd changes in striatal DA levels or behavioural impairment, even in the presence of loss of DA soma (Thiruchelvam et al., 2003). It is conceivable that compensatory/buffer downstream processes provoked by soma loss, variations in experimental design (e.g. route of administration, dosing regimen, sacri!ce interval, striatal subregions tested, age of mice) can possibly contribute to some of the inconsistency observed across studies (Rojo et al., 2007; Prasad et al., 2009; Kang et al., 2010; Rappold et al., 2010).
• The effects on nigral dopaminergic neurons appear to be speci!c (Tieu et al., 2011). However, damage in dopaminergic cell bodies and terminal has not been consistently observed (Thiruchelvam et al., 2000b; Cicchetti et al., 2005). In addition, even in studies in which a loss of nigral dopaminergic neurons is detected, PQ does not have an effect on striatal dopamine level (Thiruchelvam et al., 2000b; McCormack et al., 2002). This lack of dopamine reduction might be related to the compensatory upregulation of tyrosine hydroxylase activity in the striatum after PQ injection (Thiruchelvam et al., 2000b; McCormack et al., 2002; Ossowska et al., 2005; Tieu 2011).
• The repeat dose administration of 10 mg/kg i.p. is likely representing the maximum tolerated dose of the chemical stressor. The observed movement disorders can, at least in part, come from systemic illness and the contribution of systemic pathological changes to the observed movement disorders cannot ruled out (Cicchetti et al., 2005).
• In-vivo, experimental reproducibility of the estimated mean number of TH positive neurons in the SNpc or TH positive axons and terminals in the striatum is weak and representing an uncertainty. In particular, Breckenridge et al. (2013) and Smyne et al. (2016) conducted in vivoexperiments where the administration of the chemical stressor Paraquat at an expected neurotoxic dose and treatment schedule showed no effect. The experiments were conductedfollowing a very thorough and comprehensive protocol. In addition, Smyne et al. (2016), conducted a systematic review of the published literature that has evaluated the effects of paraquat on the SNpc and striatum in male mice. In order to evaluate potential sources of variability, multiple information was extracted and evaluated. A number of experimental limitations were identi!ed which can reduce the strength of positive outcomes observed with paraquat. Nevertheless, some positive studies could not be dismissed and the differences in host susceptibility (e.g. dose, timing of treatment, species or strain, age of animal, iron accumulation, strain speci!c gene ontology) can explain contradictory effects observed following treatment with the chemical stressor paraquat in mice.

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