Work

The parkinsonian toxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP): a technical review of its utility and safety

Year: 2001

Type: article

Abstract: Parkinson's disease (PD) is a common disabling neurodegenerative disorder the cardinal clinical features of which include tremor, rigidity and slowness of movement (Fahn and Przedborski 2000). These symptoms are attributed mainly to a profound reduction of dopamine in the striatum due to a dramatic loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (Fahn and Przedborski 2000). Thus far, both the cause and the mechanisms of PD remain unknown. Over the years, investigators have used experimental models of PD produced by several compounds such as reserpine, 6-hydroxydopamine, methamphetamine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to provide insights into the mechanisms responsible for the demise of dopaminergic neurons in PD. To this end, MPTP has emerged unquestionably as a popular tool for inducing a model of PD in a variety of animal species including monkeys, rodents, cats, and pigs (Kopin and Markey 1988). The sensitivity to MPTP and therefore its ability to induce parkinsonism closely follows the phylogenetic tree where the species most closely related to humans are the most vulnerable to this neurotoxin. Due to the significant neurotoxicity of MPTP, it is important that researchers appreciate the potential hazards of this toxin. Given this, the purpose of this review is to inform the researcher of the hazardous nature of MPTP and to provide guidance for its safe handling and use. MPTP is a by-product of the chemical synthesis of a meperidine analog with potent heroin-like effects. MPTP can induce a parkinsonian syndrome in humans almost indistinguishable from PD (Langston and Irwin 1986). Recognition of MPTP as a neurotoxin occured early in 1982, when several young drug addicts mysteriously developed a profound parkinsonian syndrome after the intravenous use of street preparations of meperidine analogs which, unknown to anyone, were contaminated with MPTP (Langston et al. 1983). In humans and non-human primates, depending on the regimen used, MPTP can produce an irreversible and severe parkinsonian syndrome that replicates almost all of the features of PD, including tremor, rigidity, slowness of movement, postural instability, and even freezing; in non-human primates, a resting tremor characteristic of PD has only been demonstrated convincingly in the African green monkey (Tetrud et al. 1986). The responses, as well as the complications, to traditional antiparkinsonian therapies are virtually identical to those seen in PD. It is believed that in PD the neurodegenerative process occurs over several years, while the most active phase of neurodegeneration is completed within a few days following MPTP administration (Langston 1987; Jackson-Lewis et al. 1995). However, recent data suggest that, following the main phase of neuronal death, MPTP-induced neurodegeneration may continue to progress 'silently' over several decades, at least in humans intoxicated with MPTP (Vingerhoets et al. 1994; Langston et al. 1999). Except for four cases (Davis et al. 1979; Langston et al. 1999), no human pathological material has been available for studies and thus, the comparison between PD and the MPTP model is largely limited to primates (Forno et al. 1993). Neuropathological data show that MPTP administration causes damage to the nigrostriatal dopaminergic pathway identical to that seen in PD (Agid et al. 1987), yet there is a resemblance that goes beyond the loss of SNpc dopaminergic neurons. Like PD, MPTP causes greater loss of dopaminergic neurons in SNpc than in ventral tegmental area (Seniuk et al. 1990; Muthane et al. 1994) and, at least in monkeys treated with low doses of MPTP but not in humans, greater degeneration of dopaminergic nerve terminals in the putamen than in the caudate nucleus (Moratalla et al. 1992; Snow et al. 2000). However, two typical neuropathologic features of PD have, until now, been lacking in the MPTP model. First, except for SNpc, pigmented nuclei such as locus coeruleus have been spared, according to most published reports. Second, the eosinophilic intraneuronal inclusions, called Lewy bodies, so characteristic of PD, thus far, have not been convincingly observed in MPTP-induced parkinsonism (Forno et al. 1993), although, in MPTP-injected monkeys, intraneuronal inclusions reminiscent of Lewy bodies have been described (Forno et al. 1986). To date, the most frequently used animals for MPTP studies are monkeys, mice and rats. The administration of MPTP through a number of different routes using different dosing regimens has led to the development of several distinct models, each characterized by some unique behavioral and/or biochemical features. The manner in which these models were developed is based on the concept of delivering MPTP in a fashion that creates the most severe and stable form of SNpc damage with the least number of undesirable consequences such as acute death, dehydration and malnutrition. Although MPTP can be given by a number of different routes, including gavage and stereotaxic injection into the brain, the most common, reliable, and reproducible lesion is provided by its systemic administration (i.e. subcutaneous, intravenous, intraperitoneal or intramuscular). The most commonly used regimens in monkeys are the multiple intraperitoneal or intramuscular injections and the intracarotid infusion of MPTP (Petzinger and Langston 1998). The former is easy to perform and produces a bilateral parkinsonian syndrome. However, often the monkey exhibits a generalized parkinsonian syndrome so severe that chronic administration of levodopa is required to enable the animal to eat and drink adequately (Petzinger and Langston 1998). On the other hand, the unilateral intracarotid infusion is technically more difficult, but causes symptoms mainly on one side (Bankiewicz et al. 1986; Przedborski et al. 1991), which enables the monkey to maintain normal nutrition and hydration without the use levodopa. For many years monkeys were mainly, if not exclusively, treated with harsh regimens of MPTP to produce an acute and severe dopaminergic neurodegeneration (Petzinger and Langston 1998). More recently, several investigators have treated monkeys with low doses of MPTP (e.g. 0.05 mg/kg 2–3-times per week) for a prolonged period of time (i.e. weeks to months) in an attempt to better model the slow neurodegenerative process of PD (Schneider and Roeltgen 1993; Bezard et al. 1997; Schneider et al. 1999). While both the acute and the chronic MPTP-monkey models are appropriate for the testing of experimental therapies aimed at alleviating PD symptoms, it is the chronic model that is, presumably, the most suitable for the testing of neuroprotective strategies. In addition to monkeys, many other mammalian species are also susceptible to MPTP (Kopin and Markey 1988; Heikkila et al. 1989; Przedborski et al. 2000). Mice have become the most commonly used species for both technical and financial reasons. However, several problems need to be emphasized. First, mice are much less sensitive to MPTP than monkeys; thus, much higher doses are required to produce significant SNpc damage in this animal species, presenting a far greater hazardous situation. Second, in contrast to the situation in monkeys, mice treated with MPTP do not develop parkinsonism. Third, the magnitude of nigrostriatal damage depends on the dose and dosing schedule (Sonsalla and Heikkila 1986). The use of MPTP in rats presents an interesting situation (Kopin and Markey 1988). For instance, rats injected with mg/kg doses of MPTP comparable to those used in mice do not exhibit any significant dopaminergic neurodegeneration (Giovanni et al. 1994a; Giovanni et al. 1994b). Conversely, rats injected with much higher doses of MPTP do exhibit significant dopaminergic neurodegeneration (Giovanni et al. 1994a; Giovanni et al. 1994b) although, at these high doses, rats have to be pretreated with guanethidine to prevent dramatic peripheral catecholamine release and extensive mortality (Giovanni et al. 1994a). These findings indicate that rats are relatively insensitive to MPTP, but regardless of this drawback, rats continue to be used often in MPTP studies (Storey et al. 1992; Giovanni et al. 1994a; Giovanni et al. 1994b; Staal and Sonsalla 2000; Staal et al. 2000). In rats, the systemic administration of MPTP is rarely used and the vast majority of studies involve the stereotaxic infusion of MPTP's toxic metabolite, 1-methyl-4-phenylpyridinium (MPP+) (Storey et al. 1992; Giovanni et al. 1994a; Giovanni et al. 1994b; Staal and Sonsalla 2000; Staal et al. 2000). Several factors influence the reproducibility of the lesion in monkeys, rats, and mice. However, to our knowledge, the extensive and systematic assessment of these factors has only been done in mice, and can be found in the following references (Heikkila et al. 1989; Giovanni et al. 1991; Giovanni et al. 1994a; Giovanni et al. 1994b; Miller et al. 1998; Hamre et al. 1999; Staal and Sonsalla 2000), the highlights of which can be summarized as follows: different strains of mice (and even within a given strain obtained from different vendors) can exhibit strikingly distinct sensitivity to MPTP. This differential sensitivity acts in an autosomal dominant fashion (Hamre et al. 1999). Gender, age, and body weight are also factors that modulate MPTP sensitivity as well as reproducibility of the lesion, in that female mice are less sensitive and exhibit more variability in the extent of damage than males, as do mice younger than 8 weeks and lighter than 25 g. From our experience, optimal reproducibility in MPTP neurotoxicity is obtained using male C57 BL/6 mice 8–10 weeks of age and 25–30 g in weight. Also of importance is that, following MPTP administration, some mice will die within the first 48 h postinjection; note that C57 BL/6 mice from different vendors exhibit dramatically different magnitudes of acute lethality, ranging from 5% to 90%. This common issue is unlikely related to a toxic effect in the central nervous system but rather toxicity to the peripheral nervous and cardiovascular systems. Although, to our knowledge, this possibility has never been formally studied, we believe that, following acute MPTP administration, mice develop fatal alterations in heart rate and blood pressure. Moreover, MPTP intoxication causes a transient drop in body temperature, which not only can modulate the extent of dopaminergic damage (Moy et al. 1998), but can also contribute to acute lethality. Death rate can be reduced by maintaining the body temperature of the injected mice using a temperature-controlled warming pad (do not use a lamp, which can kill mice by overheating them as there is no control of the temperature). MPTP has a complex multistep metabolism (Tipton and Singer 1993; Przedborski et al. 2000). It is highly lipophilic, and freely and rapidly crosses the blood–brain barrier. Within a minute after MPTP injection, levels of the toxin are detectable in the brain (Markey et al. 1984). Once in the brain, MPTP is metabolized to 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+) by the enzyme monoamine oxidase B (MAO-B) in non-dopaminergic cells. Then MPDP+ is oxidized to the active MPTP metabolite, MPP+, which is then released into the extracellular space, where it is taken up by the dopamine transporter and is concentrated within dopaminergic neurons, where it exerts its toxic effects. The essential role of these different metabolic steps in MPTP-induced neurotoxicity and the fact that MPP+ is the actual culprit are demonstrated by the following observations: (1) pretreatment with MAO-B inhibitors such as deprenyl prevents MPTP biotransformation to MPP+ and blocks dopaminergic toxicity (Heikkila et al. 1984; Markey et al. 1984); (2) pretreatment with dopamine uptake inhibitors (e.g. mazindol) prevents MPP+ entry into dopaminergic neurons and also blocks dopaminergic toxicity (Javitch et al. 1985), at least in mice; and (3) striatal MPP+ content correlates linearly with dopaminergic toxicity in mice (Giovanni et al. 1991). Knowing where MPTP and its toxic metabolite, MPP+, accumulate both inside and outside of the body of the injected animal following MPTP administration is germane to the formulation of any set of standard practices for the safe use of MPTP. Following MPTP administration to both mice and monkeys, only the interior surfaces of the cage, the surfaces that the animals and/or their excreta could physically touch, including food and drinking bottle, are contaminated with MPTP and its metabolites (Yang et al. 1988). Conversely, no evidence of contamination is found outside of the cage or on the outside surrounding surfaces (Yang et al. 1988). At two days postinjection, 70% of the total injected dose of MPTP and its metabolites is recovered from the inside cage-wash, urine and feces, of which about 15% in mice and 2% in monkeys is unmetabolized MPTP, while the rest is due to MPTP metabolites, such as MPP+. Moreover, it appears that the excretion of unmetabolized MPTP occurs mainly during the first day postinjection, while mainly MPTP metabolites are excreted up to 3 days postinjection (Yang et al. 1988). There is no evidence either in mice or in monkeys that MPTP and its metabolites are still being excreted after 3 days post MPTP administration. Although high concentrations of MPTP are found in the bile, the main route of MPTP excretion is the urine (Johannessen et al. 1986). MPTP in urine will likely be ionized and not volatile, and be well absorbed by the animal bedding. Also, less than 0.01% of the total injected dose of MPTP is detected as volatile MPTP, which probably originates from the animals exhaling MPTP or from vapors from contaminated urine (Yang et al. 1988). One day after an injection of radiolabeled MPTP to mice, most of the radioactivity is localized in the brain and the adrenal gland, while all other organs contain 50–75% lower amounts of radioactivity (Johannessen et al. 1986). Analysis of the radioactive species recovered from different organs and body fluids such as bile, urine, blood, and CSF demonstrates variable amounts of unmetabolized MPTP soon after injection, but by 12–24 h postinjection, essentially all of the radioactivity corresponds to MPP+ (Markey et al. 1984; Johannessen et al. 1986). From the above, it appears that the potential risks of exposure to MPTP are through direct contact with the animal, the animal cage inner surfaces, and its bedding material. There is minimal risk from exposure due to airborne or vapor-borne forms of MPTP. Although safety procedures, as outlined below, must always be followed, the period of maximal risk of MPTP contamination is from the moment of injection to the time that MPTP or its metabolites are no longer found in the excreta of treated animals; as a precautionary measure, we recommend extending the period of high-risk from 3 days to 5 days post-MPTP injection. Prior to discussing MPTP preparation, injection and animal experimentation, it is necessary to discuss the issues of the recommended facility and personal protective equipment (PPE). As a rule, only investigators and/or staff members who are trained in handling hazardous agents and who are familiar with MPTP safety procedures and practices should prepare and administer MPTP, and monitor the animals during the high-risk period (i.e. 5 days post-MPTP injection). Of note, any staff member who undertakes these tasks should give informed consent and not be coerced into taking on MPTP-related duties. Moreover, it is strongly recommended that all aspects of the MPTP experiment, including storage and solution preparation, take place in a dedicated procedure room (for small animals) or area within the animal room (for large animals), and not in a regular laboratory. For personal safety, when using MPTP, researchers are required to wear the PPE described below, during the preparation of the MPTP solution, the injection period, and 5 days postinjection. Thereafter, regular laboratory attire as required to handle animals is sufficient. It is important to emphasize that in laboratories committed to MPTP research, one cannot exclude that exposure to even trace amounts of MPTP over many years of the same investigator and/or staff member may have negative consequences. This is one more reason why a heightened standard of protection must be implemented for any individual involved in MPTP experiments. All MPTP experiments including preparation of solutions must be performed in a procedure or animal room under negative-pressure because aerosols from MPTP and its metabolites can be generated from bedding, excreta and animal fur. All animals should be acclimated to the room for 4–7 days prior to any MPTP experiment to allow for monoamine stabilization before MPTP injection since monoamine level alterations may affect intragroup lesion reproducibility. The procedure or animal room should have a 12-h light-dark cycle, a bench with a working area, a sink, and be temperature-controlled. For small animals like mice, it should also be equipped with an animal rack to hold all of the cages and a fume hood. All furniture should be of stainless steel or of any material, except wood, that is acid-resistant and washable. All working surfaces including the fume hood and animal racks should be covered with materials that are absorbent on the face-up side and non-absorbent on the face-down side. The entire floor of the procedure room or working area in the animal room for large animals should be covered with plastic-backed absorbent sheets. A warning sign clearly stating 'Danger! MPTP Neurotoxin Use Area – Entry Restricted' must be posted on the outside of the procedure or animal room door. The room must be locked at all times and the animal care staff informed of the ongoing use of MPTP and its dangers. They must also be informed that this room is off limits unless allowed to enter by the responsible investigator. This procedure room or designated procedure area should be completely equipped with all of the necessary supplies for the MPTP experiments. It should also contain a sharps disposal container clearly labeled as hazardous waste, a container lined with a hazardous disposal for animal absorbent for animals and MPTP, with solution in a personal protective equipment and deprenyl an MAO-B for exposure to MPTP. It is that the material safety data for MPTP, which is by the be in the in the room or area, there should be no need to during the injection PPE must be during all procedures MPTP, including during the 5 days post-MPTP injection. The PPE is far more important when mice than monkeys as mice higher doses of MPTP. The PPE of a with an and of a and non-absorbent material that is and protection from airborne This should be easy to into and to after one For of with and and an hood can be A with that is to the individual is for and a with that is by the of and for protection that is to the individual using the can also be The is and should be with solution then with after each must be of in the hazardous and under the PPE All the PPE attire can be obtained from a large laboratory The of and safety in any where MPTP is to be used must be for guidance in PPE attire for use with MPTP. For small animals such as mice, cages and are strongly recommended as of without bedding cages with is recommended to both room contamination and contamination of other animal cages should be on the animal rack in the procedure room prior to and during the period post-MPTP injection. All injections must be performed in the fume hood in the procedure For large animals such as monkeys, cages should be The of the cage and the drop must be lined with plastic-backed absorbent MPTP can be from several do not use MPTP as the but only as the or other MPTP storage and handling must be to the procedure room or designated area within the animal the use of large concentrated and handling of MPTP and never MPTP solutions or of MPTP outside of the dedicated MPTP may be in small of or in of MPTP must be until used and at room temperature in a container within a This container should be in a locked with a This must be to a in the procedure room or investigators trained in the handling of MPTP should perform the Use of will handling problems that from the of It is strongly recommended that a dedicated to MPTP be in the procedure Prior to MPTP the area with with solution to the risk of airborne MPTP To the risk of MPTP the procedure described by et al. is a safe a small container (e.g. small with a take the container and place an of MPTP in and container with then to give container and all other with of all in a hazardous if a given experiment a total dose of less than or of MPTP, then it is not to the and the but to the of to the or It must be that this MPTP solution has to be used in one day and the since MPTP in solution at room prior to the used MPTP a of solution to the of MPTP solution into the then the as found that MPTP solution at its as MPTP solution appears stable up to However, unless one has a dedicated for MPTP other issues such as laboratory safety will and that even without the negative of and of MPTP solution on its should be injected only with solutions of MPTP by either through a or by the in or not MPTP as this will the and may to exposure from As above, a number of different injection regimens have been used to produce the MPTP These are based on a number of including experimental of lesion, and species As mice, which greater amounts of MPTP to produce can be injected either or or multiple injection, and with a of the regimen used, it is recommended that all MPTP injections to mice be performed in a fume hood. from which MPTP is should have a or be covered with to potential aerosols and and to on the frequently during the of and at the of the injection This will prevent any contamination of the PPE and the possibility of contamination of On the day of or on the before the experiment, all animals are and a before the injection MPTP solution should be to the working animal injection, care must be taken to to animal will this For injection, place the cage in the fume hood and when hold the animal so that any urine will into the cage and not on the surrounding since mice, when to urine which can contain significant amounts of MPTP (Yang et al. 1988). the is not so as to cause of the injected MPTP from the animals such as monkeys must be in for injection. It is not to large animals in a fume hood. injection for or solution and with a small pad with do not or from the with solution and then place the with in a sharps container to be of as At the of the injection the MPTP solution must be with an of solution as described The potential for exposure to MPTP and its metabolites is from contaminated bedding and following MPTP injection and during the period that MPTP or its metabolites are likely to be in the excreta of treated when handling cages and their it is important that the PPE be cages contaminated bedding should be with solution and then be into a and for using bedding should also be with solution, then in the and of as after cages and with solution for then with and with cages may then be to central cage The absorbent material that covered the rack surfaces should be with solution, allow to for and then of as hazardous For large animal plastic-backed absorbent that the cage and drop with solution, allow to for then and place them in the used with This to be done on a cages and with solution, then with and with The procedure described that MPTP-injected animals remain in the same cage for 5 days postinjection and should only after the 5 days postinjection In the of prolonged MPTP exposure (i.e. weeks to while the procedure room or area will remain the period the days postinjection for mice, only cage a following the procedure described and, for monkeys, it is to monkeys to cages other and to handle the cages as described in the procedure room or area should be with should be and of as hazardous animal care can be days post MPTP injection and the procedure room or area has been by the responsible investigator and/or staff risk of exposure to MPTP or MPP+ may when animals are for up to 5 days following MPTP administration. this period, mice should be in the fume hood and the appropriate PPE by the researcher during blood and All working surfaces are lined with plastic-backed absorbent which should be if with body is the of for small animals in MPTP care should be taken to prevent blood and urine and should be are on an covered with and on

Source: Journal of Neurochemistry

DOI: 10.1046/j.1471-4159.2001.00183.x