A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing >95% loss of striatal dopamine and 90.63±-4.02 % ipsiversive rotational bias towards the side of the lesion.
The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson’s disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate >80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes >95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.
1. Housing and preparation of mice
2. Preparation of drugs for surgery
A premedication of desipramine and pargyline is usually administered to rodents prior to injection of 6-hydroxydopamine (6-OHDA) to increase the selectivity and efficacy of 6-OHDA-induced lesions. The noradrenaline / 5HT uptake inhibitor, desipramine decreases 6-OHDA-induced noradrenaline and 5HT depletion22, whereas the monoamine oxidase inhibitor, pargyline enhances the sensitivity of dopaminergic terminals to 6-OHDA, by reducing extrasynaptic breakdown of 6-hydroxydopamine31.
3. Setting up the surgical apparatus
Surgical tools must be sterilised using an autoclave prior to surgery. Between each operation, surgical tools must be sterilised in 95% ethanol followed by heating to 250°C for 2 minutes. All experiments must be performed in accordance with the guidelines of the appropriate Animal Care Committees of the affiliated institution and country.
4. Unilateral 6-OHDA lesion surgery
5. Post-operative care of 6-OHDA-lesioned animals
6. Parkinsonian assessment
To estimate the extent of the lesion, behavioural assessment was performed 14-21 days following 6-OHDA-lesion surgery, when the amount of dopamine depletion is maximal and stable26.
7. Determination of striatal dopamine content by HPLC
8. Representative Results
Fifteen to twenty one days following 6-OHDA-lesion surgery, when the amount of cell death caused by the neurotoxin has reached completion26, measurement of spontaneous 360° rotations following administration of saline (i.p.)28 can be used to assess the success of 6-OHDA-lesioned (Figure 2). This method of behavioural assessment is simple and quick, and avoids potential priming effects caused by dopaminergic agents such as amphetamine or apomorphine challenges. Furthermore, spontaneous rotation assessment is a better predictor of animals with <95% dopamine depletion compared to forelimb paw placement test27. By correlating striatal dopamine levels from striatal samples (prepared upon completion of behavioural studies using HPLC) with percentage of spontaneous ipsiversive rotations in each animal, we have found that animals which exhibit 70% or more ipsiversive rotations towards the 6-OHDA lesion have lost >95% striatal dopamine (Figure 3)27. Following partial 6-OHDA-induced lesions (59.44±17.20) (Figure 3) of the medial forebrain bundle, there was no rotational bias between ipsiversive versus contraversive sides when measuring spontaneous rotations (40.91±8.01) (Figure 2). Thus, as has been found following 6-OHDA administration to the MFB in rats, it is possible to cause a profound loss of dopaminergic nigro-striatal neurons, and saline administration (i.p.) is an accurate method for screening the extent of 6-OHDA lesion in these animals.
Figure 1. Schematic diagram to demonstrate assembly of the injection needle apparatus for 6-OHDA-lesion surgeries.
Figure 2. Assessment of rotational behaviour in sham-operated and 6-OHDA lesioned mice. Spontaneous rotations in sham-operated and 6-OHDA-lesioned mice. Data are presented as mean percentage ipsiversive rotations ± SEM, where net contraversive and ipsiversive rotations are 100%. Open bars: sham-operated animals; Grey bars: 6-OHDA-lesioned animals with >95% dopamine loss; Black bars: animals that underwent 6-OHDA-lesion surgery that were partially lesioned. *** P<0.001 compared to sham-operated animals. One-way ANOVA followed by Dunn’s multiple comparison test (sham-operated: n=17; 6-OHDA-lesioned (>95%): n=23; partial 6-OHDA-lesion: n=3).
Figure 3. Assessment of striatal dopamine levels in sham-operated and 6-OHDA lesioned mice using HPLC. Striatal dopamine content was determined in the operated and unoperated hemisphere of sham and 6-OHDA-lesioned animals. Data are presented as mean percentage ± SEM of striatal dopamine levels in the unoperated striatum. Open bars: sham-operated animals; Grey bars: 6-OHDA-lesioned animals with >95% dopamine loss; Black bars: animals that underwent 6-OHDA-lesion surgery that were partially lesioned. *** P<0.001, ** P<0.01 compared to sham-operated animals. One-way ANOVA followed by Dunn’s multiple comparison test (sham-operated: n=17, 6-OHDA-lesioned (>95%): n=23, partial 6-OHDA-lesion: n=3).
Figure 4. Assessment of tyrosine hydroxylase immunoreactivity in the SNc in sham-operated and 6-OHDA lesioned mice. As a marker of dopamine cell loss in the substantia nigra pars compacta (SNc), loss of tyrosine hydroxylase (TH) positive immunohistochemisty in the operated and unoperated hemisphere of sham and 6-OHDA-lesioned animals was determined as described in Thiele et al. In Press. Data are presented as mean percentage ± SEM of TH positive cells in the unoperated striatum. Open bars: sham-operated animals; Grey bars: 6-OHDA-lesioned animals with >95% dopamine loss; Black bars: animals that underwent 6-OHDA-lesion surgery that were partially lesioned. *** P<0.001, ** P<0.01 compared to sham-operated animals. One-way ANOVA followed by Dunn’s multiple comparison test (sham-operated: n=17, 6-OHDA-lesioned (>95%): n=23, partial 6-OHDA-lesion: n=3).
This protocol describes a method for the generation of a stable unilateral 6-OHDA-lesioned mouse model of Parkinson’s disease, which is extremely reproducible, with a high lesion success rate, and a low mortality rate. The success of 6-OHDA lesion surgery can be easily estimated by the measurement of spontaneous rotational behaviour with >70% ipsiversive rotations indicative of >95% dopamine depletion in the lesioned striatum27. Quantification of striatal dopamine levels is the most accurate measurement of the extent of striatal dopamine depletion compared to quantificiation of the number TH-positive cells in the substantia nigra pars compacta, as it provides a direct measurement of striatal dopamine levels28. Given that this protocol reproduces the site of injection (MFB) and length of lesion development (21 days) of the well characterised unilateral 6-OHDA-lesioned rat model of Parkinson’s disease1,4, it can be assumed that this mouse model, as with the rat model, recapitulates the changes in pharmacology and brain circuitry observed in patients with Parkinson’s disease. Thus, this method provides a means of generating a symptomatic model of Parkinson’s disease in transgenic mice. Given that transgenic mice are extremely powerful tools for allowing us to understand the role of molecules and proteins in vivo at the molecular, through to the cellular and whole tissue level, combining these two techniques will prove extremely useful in further delineating the molecular and cellular mechanisms underlying generation of symptoms of Parkinson’s disease.
Cenci has previously described a similar method for generating a unilateral 6-OHDA-lesioned mouse of Parkinson’s disease, however, this was associated with a high mortality rate, with only 14% of animals surviving more than 21 days post surgery following injection of 6-OHDA into the MFB11. There are several possible explanations for the lack of mortality in the protocol described here. Firstly, the stereotaxic co-ordinates chosen in this protocol allow the injection needle to completely avoid the hypothalamus. Since the mouse hypothalamus is so small, any damage to this structure may affect the eating and drinking centres of the mouse, causing the animal to lose weight and become dehydrated, and ultimately die. Secondly, in the protocol described here, the same concentration of 6-OHDA (3mg) was utilised as in the study by Lundblad and colleagues11, however, it was administered in a smaller volume (0.2ml compared to 1ml) with a slower infusion rate (0.1ml/min compared to 0.5ml/min). The smaller volume and slower infusion rate ensure minimal damage to structures surrounding the MFB. Finally the diameter of the injection needle is significantly smaller (0.33 vs 50mm), which will minimise damage to brain structure as the needle travels ventrally through the brain.
Cenci has also described another method for generating unilateral 6-OHDA-lesions, with 6-OHDA being injected into the striatum11. As has been described following striatal injection of 6-OHDA in rats, this results in a lower level of dopamine loss, which is more variable between mice.
A final consideration that must be taken into account is the potential effect of strain of mice. It has been well documented the strain has a dramatic effect on success of dopamine depletion following systemic administration of MPTP, which also causes selective cell death of the dopaminergic nigro-striatal pathway29. While this difference between species does not seem to occur in rats following 6-OHDA administration, it is possible that it may be a factor in determining the success of 6-OHDA lesion in mice.
The authors have nothing to disclose.
This work was supported by the Department of Foreign Affairs and International Trade (Government of Canada), University of Toronto Connaught Fund, the Canadian Foundation for Innovation, NSERC, the Krembil Foundation and the Cure Parkinson’s Trust.
Name of the reagent | Company | Catalogue number | Comments (optional) |
desipramine HCl | Sigma-Aldrich, Oakville, ON, Canada | D125 | 25mg/kg |
pargyline HCl | Sigma-Aldrich, Oakville, ON, Canada | P8013 | 5mg/kg |
6-OHDA HBr | Sigma-Aldrich, Oakville, ON, Canada | H116 | 3mg / mouse |
stereotaxic Frame | Kopf Instruments, Tujunga, CA, USA | Model 900 | |
mouse ear cups | Kopf Instruments, Tujunga, CA, USA | Model 921 Zygoma Ear Cups | |
mouse incisor bar | Kopf Instruments, Tujunga, CA, USA | Model 923B | |
mouse anaesthesia mask | Kopf Instruments, Tujunga, CA, USA | Model 923B | |
priming kit (containing 250ml syringe) | Hamilton Company, Reno, NV, USA | PRMKIT 81120 | |
RN compression fitting kit (1 mm) | Hamilton Company, Reno, NV, USA | 55750-01 | |
PEEK tubing from RN compression fitting kit< (1/16th inch) | Hamilton Company, Reno, NV, USA | 55751-01 | |
dual small hub RN Coupler | Hamilton Company, Reno, NV, USA | 55752-01 | |
luer to small hub RN adaptor | Hamilton Company, Reno, NV, USA | 55753-01 | |
1ml 25S syringe model 7001KH | Hamilton Company, Reno, NV, USA | 80100 | |
*33G removable needle (RN) pack of 6. . Custom 1 inch with 45<° bevel | Hamilton Company, Reno, NV, USA | 7803-05 | |
Scissors | Fine Science Tools, Vancouver, BC, Canada. | 14084-08 | |
Scalpel | Fine Science Tools, Vancouver, BC, Canada | 10003-12 | |
Scalpel blades | Fine Science Tools, Vancouver, BC, Canada | 10035-20 | |
Forcep | Fine Science Tools, Vancouver, BC, Canada | 11608-15 | |
Hemostats | Fine Science Tools, Vancouver, BC, Canada. | 13004-14 | |
Isoflurane | Abbot | 02241315 | 2-3% |
Suters (Vicryl 4.0) | Syneture | SS-683 | |
Steriliser | Fine Science Tools, Vancouver, BC, Canada | 18000-45 | |
Infusion Pump | Harvard Apparatus | PhD 22/2000 | |
Needles (27G) | Becton Dickinson | 305109 | |
Needles (25G) | Becton Dickinson | 305127 | |
Syringes (1ml) | BD syringe | 309692 | |
Anaesthesia trolley | LEI medical | M2000 | |
Baytril | CDMV, St. hyacinthe, QC | 102207 | |
Lidocaine | CDMV, St. hyacinthe, QC | 3914 | |
Betadine solution | CDMV, St. hyacinthe, QC | 19955 |