6-OHDA Lesion Models of Parkinson’s Disease in the Rat

267Emma L. Lane and Stephen B. Dunnett (eds.), Animal Models of Movement Disorders: volume I , Neuromethods, vol. 61,DOI 10.1007/978-1-61779-298-4_13, © Springer Science+Business Media, LLC 2011Chapter 136-OHDA Lesion Models of Parkinson’s Disease in the RatEduardo M. Torres and Stephen B. DunnettAbstractThe 6-hydroxydopamine (6-OHDA) lesion of the rat nigrostriatal pathway is the most widely used animal model of Parkinson’s disease. 6-OHDA is a highly specific neurotoxin which targets catecholamine neurones via the dopamine active transporter (DAT). When injected stereotaxically into the brain, either into the median forebrain bundle (MFB) or into the neostriatum, it causes extensive, irreversible loss of dopamine neurones in the ventral midbrain. The corresponding loss of dopamine innervation in target areas is associated with a range of long-term, behavioural deficits that form the target of experimental therapies, aimed at protecting or restoring dopaminergic deficits. In this chapter, the two most widely used 6-OHDA lesion protocols are described: (1) The MFB lesion that results in >97% unilateral depletion of dopamine neurones, principally in the ipsilateral striatum and nucleus accumbens. (2) The unilateral striatal lesion resulting in partial dopamine denervation of the striatum only. In vivo assessment of both lesion types by drug-induced rotation is also covered.Key words: 6-Hydroxydopamine, Dopamine lesion, Parkinson’s, Amphetamine rotation, Apomorphine rotationThe specific neurotoxic effects of 6-OHDA rely on its structural similarity to dopamine, whereby dopamine reuptake mechanisms, principally the dopamine active transporter (DAT), selectively take up the neurotoxin, which becomes concentrated within dopamine neurones. Subsequent metabolism of the neurotoxin results in the production of toxic free-radicals within the cell, causing rapid dysfunction and eventual cell death. As 6-OHDA does not cross the blood–brain barrier, it must be injected stereotaxically into the brain. Intracerebral injection of 6-OHDA to induce lesions of the dopamine system in the brain dates back more than 40 years to work done by Urban Ungerstedt, who injected the neurotoxin1. Introduction268 E.M. Torres and S.B. Dunnettdirectly into the rat substantia nigra compacta (SNc) to induce“anterograde degeneration of the whole nigrostriatal dopaminesystem” (1). The modern version of the lesion targets the medianforebrain bundle (MFB), the anterior efferent from the dopamin-ergic substantia nigra compacta, and ventral tegmental area (2).Injection of 6-OHDA into the MFB results in rapid uptake ofthe toxin into dopamine neurones, and a near complete loss of thedopamine neurones in these nuclei. There is extensive dopaminedepletion of target areas on the side of injection, including thestriatum, nucleus accumbens, septum, prefrontal cortex and olfactorybulb. Unfortunately, bilateral MFB lesions produce severe akinesia,aphagia and adipsia (3) and are unsuitable for most experiments.By contrast, unilaterally lesioned animals are outwardly normal,and capable of eating and drinking normally, and for this reasonunilateral lesions are generally favoured. A large number of similarstereotaxic coordinates for injection into the MFB are available inthe scientific literature. The recent literature also contains coordi-nates for double injection strategies aimed at the MFB, but it is theauthors’ experience that a well targeted single injection is considerablymore straightforward, and, provided that the injection is targetedaccurately, more than capable of causing near complete lesionsof the MFB.Partial lesions of the dopamine system may also be achieved byinjection of 6-OHDA directly into the striatum. Using thisapproach results in a loss of dopamine neurones in the substantianigra and corresponding dopamine denervation in the striatum. Butdopamine innervation to other brain areas, notably the nucleusaccumbens, is spared. Additionally, using the striatal route of toxinadministration, the extent of the lesion may be varied, accordingto the dose of 6-OHDA injected, and so-called “partial lesions”may be achieved (4, 5).The behavioural deficits produced by 6-OHDA lesions havebeen described previously and are not discussed in detail here. (seeFleming and Schallert in this volume). However, as no lesion pro-tocol is 100% efficient, lesioned animals require behavioural testingin order to determine whether or not the lesion has been successful.The standard method of assessment is to observe and record turn-ing behaviour under the influence of dopaminergic stimulantdrugs. In rats with unilateral lesions, injections of pharmacologi-cal doses of amphetamine or apomorphine induce a striking turn-ing behaviour known as “rotation” and which correlates closelywith the extent of dopamine denervation (6). Amphetamine stim-ulates dopamine release and blocks reuptake from dopaminergicterminals in the intact striatum to a greater extent than on thelesioned side of the brain, resulting in a profound motor asymme-try. Together with the increase in activity caused by the drug, thiscauses the animal to adopt a classic nose-to-tail posture and to rotatevigorously towards the side of the lesion (ipsilateral). Apomorphine,26913 6-OHDA Lesion Models of Parkinson’s Disease in the Rat on the other hand, stimulates supersensitive receptors in the lesioned striatum in preference to the normal receptor comple-ment on the intact side, and results in rotation in the opposite direction, towards the intact (contralateral) side. (For a detailed description of rotation, see Dunnett and Torres, this volume).In the present chapter, we describe the methods to carry out unilateral dopamine-depleting lesions in the rat by injection of 6-OHDA into the forebrain, either into the MFB or into the corpus striatum, and for subsequent rotation testing using both amphet-amine and apomorphine.6-OHDA is highly toxic, and should be handled using the appropriate protective clothing (see the material safety data sheet that comes with the product for full details). The active neurotoxin is the 6-OHDA hydrobromide salt (for control experiments, the less active hydrochloride salt may be used). This can be obtained from a number of suppliers, each of which supplies a slightly different formulation. For the purposes of determining dosage, the final concentration of the neurotoxin must therefore be calculated on the weight of the free base. In solid form, 6-OHDA is sensitive to both light and temperature, and should be stored in a −20°C freezer. In solution, it oxidizes rapidly and all solvents should contain 0.01–0.1% ascorbic acid as an antioxidant. The 6-OHDA formulation supplied by Sigma-Aldrich (UK) has a molecular weight of 250.09 and contains ascorbic acid as a stabilizer. This can therefore be dissolved, without further addition of an antioxidant, using 300 mM sodium chloride (0.9% saline) solution. Sterile, nonpyrogenic saline in infusion bags is ideal.A working concentration of Sigma-Aldrich 6-OHDA is achieved by dissolving 5 mg of the formulation in 0.8 ml of 300 mM sodium chloride (sterile saline infusion solution). This gives a solution containing 30 mM 6-OHDA in 0.03% ascorbic acid and free-base weight of 6-OHDA of 5.14 mg/ml. After dissolving the toxin, quickly divide the solution into 50 m l aliquots in 1.5 ml Eppendorf tubes and store at −20°C until needed. Aliquots are stable for at least 1 month. On the day of surgery, a single aliquot is thawed quickly, and then stored on ice during surgery. After 2 h, the original aliquot should be discarded and a fresh aliquot thawed. Repeat as necessary.Amphetamine is a presynaptic dopamine agonist which activates the intact (contralateral) side of the brain to a much greater extent than the lesioned (ipsilateral) side. Crossover of striatal outputs leads to a principally attentional neglect on the contralateral side and causes the animal to rotate ipsilaterally (towards the lesion) (7).2. Materials2.1. Preparation of 6-OHDA2.2. Preparation of Dopamine Active Drugs2.2.1. Amphetamine270 E.M. Torres and S.B. DunnettSeveral different forms of amphetamine have been used in different studies, most commonly either d-amphetamine or the more potent methamphetamine. In our laboratory, we use a standard dose of 2.5 mg/kg methamphetamine hydrochloride for rotation screening of 6-OHDA lesions. Make up the drug at 2.5 mg/ml in 0.9% sterile saline and inject intraperitoneally at a dose of 1 ml/kg immediately prior to rotation testing (see below).Apomorphine is a dopamine receptor agonist which acts on both D1 and D2 receptors located primarily on the post-synaptic medium spiny projection neurons in the striatum. Following a dopamine lesion, the receptors in the lesioned striatum upregulate to compensate for the loss of dopamine, a phenomenon known as supersensitivity (8). Consequently, in a unilaterally lesioned animal, it is the lesioned side (ipsilateral) which is preferentially activated by the agonist, in particular at very low doses that are subthreshold for activating normo-sensitive receptors on the intact contralateral side of the brain. Because of the crossover of striatal output, activa-tion in the lesioned striatum drives the animal to rotate toward, the opposite (contralateral) side. Whilst doses of 0.25–1.0 mg/kg induce locomotor activation in intact rats, a dose an order of magnitude lower is sufficient to activate supersensitive receptors in the 6-OHDA lesioned striatum. In our laboratory, we use a standard dose of 0.05 mg/kg to test for agonist-induced rotation. Make up the drug at a concentration of 0.1 mg/ml in 0.1% ascorbic acid in 0.9% sterile saline, and inject subcutaneously in the neck scruff at a dose of 0.5 ml/kg. Note that this dose of apomorphine is designed to produce maximal striatal asymmetry by preferential activation of receptors on the lesioned (supersensitive) side, with minimal receptor activation on the intact side. Higher doses may be used, some workers preferring 0.25 mg/kg, but this is less effective because it involves a competition between levels of activation on the two sides, and higher doses also induce focal stereotypy responses which compete with the biased locomotor activation which underlies rotation.The preferred method for anaesthesia is by inhalation, depending on the surgical setup available. In our laboratory, we use the gaseous anaesthetic isoflurane vaporized into a 2:1 mixture of O 2:NO carrier gas delivered via an inhalation mask fixed around the incisor bar of the stereotaxic frame. Long-lasting injectables may also be used (e.g. equithesin, ketamine), but dopamine inter-acting drugs such as barbiturates should be avoided. The 6-OHDA neurotoxin is delivered using stereotaxic methods. We use a Kopf2.2.2. Apomorphine3. Methods3.1. General Surgical Procedure271 13 6-OHDA Lesion Models of Parkinson’s Disease in the Rat stereotaxic frame with adjustable nose bar height, blunt (45°) ear bars, cannula holder and drill holder. Note that although many experimenters prefer 17° ear bars, as they are easer to apply, these are more likely to cause inner ear damage, resulting in vestibular balance impair-ments and torsional body twisting, which compromises the reli-ability of subsequent rotation tests.6-OHDA is delivered via a 30 guage, stainless steel cannula connected by 0.5 m of polythene tubing (Portex internal diameter 0.28 mm, outer diameter 0.61 mm) to a 10 m l microsyringe (SGE Europe Ltd, Milton Keynes, UK or Hamilton Co., USA) (see Fig. 1). As 30 guage cannula is easily bent, this can be reinforced with an outer reinforcing sleeve of 23 guage steel tubing, either crimped or glued to the injection cannula, and allowing at least 15 mm of the 30 guage tubing free at both ends. Prior to use, the syringe, cannula and tubing must be primed with sterile, isotonic saline solution. Any air remaining in the system is liable to com-pression during injection and may prevent expulsion of the toxin from the cannula. The syringe is driven by a motorized syringe pump calibrated to deliver 1 m l/min. Note that direct injection using a suitably sized microsyringe is possible, by repeated small depressions of the plunger over 3 min to deliver 3 m l. Holes in the skull are made using a motorized, surgical drill attached to the stereotaxic frame, with a size 1/2 drill bit. Because of the small size of the hole in the skull, the cannula can be accurately positioned for injection, without the need for repositioning from bregma. If a larger drill bit is used, or if hand drilling is necessary, the needle should be zeroed using bregma and positioned using the x– y coordinates.Fig. 1. Ten-microlitre microsyringe setup for MFB lesion injections. The 30-gauge can-nula is connected to the needle of the syringe using polythene tubing. The syringe, tubing and cannula are filled with sterile infusion saline prior to use (priming).272 E.M. Torres and S.B. DunnettTable 1 gives two sets of coordinates; the first is based on Dunnett et al. (9) and is designed for use on rats over 250 g in weight. The second is based on recent modifications made by Torres et al. [38] and is recommended for rats in the 150–250 g weight range. 1. Anaesthetize the rat, and place the rat in the stereotaxic framewith the nose bar set at the appropriate height (see Table 1). 2. Shave the scalp using hair clippers, and make an incision usinga scalpel, through the skin along the midline (maximum 2.5 cm long) starting from just behind the inter-ocular line, and cutting caudally. 3. Open the incision and use the scalpel blade to pare back theunderlying connective tissue and scrape the exposed surface of the skull clean. 4. Position the tip of the drill bit directly over bregma; the zeropoint for x – y coordinates. 5. Raise the drill bit from the skull and move it to the x – y coor-dinates (see Table 1). Drill through the skull, taking care not to damage the underlying dura matter. 6. Load the cannula with 4–5 m l of 6-OHDA, first drawing asmall “spacing” air bubble (0.5–1 m l) into the cannula to prevent mixing of the neurotoxin and the priming solution. 7. Lower the cannula into the drilled hole until it is just touchingthe dura mater. If the dura matter is undamaged, the correct depth is determined when the meniscus of fluid in the drill hole descends slightly, as the dura membrane is touched by the cannula. 8. Lower the cannula into the brain, allowing the cannula topierce the dura mater to the desired depth from the dura mater. 9. Start the syringe pump and run for 3 min to deliver 3 m l ofneurotoxin. Smooth flow of the toxin without blockage of the cannula is monitored by movement of the spacing bubble in the transparent polyethylene tubing.3.2. Detailed Surgical Methodology3.2.1. Unilateral Lesion of the MFBTable 1Stereotaxic coordinates for MFB injection of 6-OHDACoordinates from bregma (mm)Nose barA =L =V =aaNegative lateral coordinates denote right-sided lesions27313 6-OHDA Lesion Models of Parkinson’s Disease in the Rat 10. Stop the pump and leave the cannula in place for a further2 min to allow fluid diffusion, before slowly retracting from the brain. 11. Purge any unused neurotoxin from the cannula and flushseveral times by repeated drawing and expulsion of clean saline solution. 12. Suture the skin and administer analgesia according to the localSOP . 13. Place animal in a heated recovery cage until fully conscious.With gaseous anaesthesia this usually takes 5–10 min, after which the rat can be returned to its home cage.The 6-OHDA has an immediate effect; recovering animals will have a postural bias towards the lesion side and rotate ipsilaterally. Checking for rotation in recovering animals is the first index of lesion success. Post surgery complications with this type of lesion are very rare but animals should be given a thorough check 24 and 48 h later.Table 2 shows coordinates for multiple injection of the toxin into the striatum. The coordinates are based on those described by Kirik et al. (4) modified so that the four injection sites are in a straight line, 1 mm apart, enabling four cannulae (each attached to a different syringe) to be used simultaneously. This requires a cannula holder like the one shown in Fig. 2 that has vertical grooves, exactly 1 mm apart, in which the cannulae are held tightly at the correct spacing. The cannulae are adjusted to the same length in the cannula holder by loosening the clamp and lowering the cannula tips onto the top surface of the ear bar before re-clamping. If a single syringe and cannula are used, drill all four holes before making the first injection.3.2.2. Partial Lesions by Striatal Injection of 6-OHDATable 2Stereotaxic coordinates for striatal injection of 6-OHDACoordinates from bregma (mm)aA =L =V =Note that, because the drill holes are evenly spaced, after the positioning and drilling of the first hole, movement between subsequent drill holes is the same: A = −0.6, L = −0.8aNegative lateral coordinates denote right-sided lesions bModified from Kirik et al. (4)274 E.M. Torres and S.B. DunnettNote that, because the drill holes are evenly spaced, after positioning and drilling the first hole, movement between subsequent drill holes is always the same: A = −0.6, L = −0.8.1. Place the rat in the frame with the nose bar set at the appropriateheight (see Table 2). 2. Shave the scalp and make an incision in the skin along the midline(maximum 2.5 cm). 3. Open the incision and clean the surface of the skull with ascalpel blade. 4. Position the tip of the drill bit over bregma.5. Move the drill bit to the x – y coordinates for the first hole (seeTable 2) and drill through the skull, taking care not to damage the underlying dura matter. 6. Repeat the process for holes 2–4.7. Load each cannula with 3–4 m l of 6-OHDA, leaving a small airbubble in each syringe setup, between the neurotoxin and the priming solution. 8. Lower the cannulae until just touching the dura matter (seeabove). 9. Lower the cannula into the brain to the maximum depth fromthe dura matter. 10. Start the syringe pump and run for 2 min, raising the cannula(e)0.5 mm after 40 s and again after 1 m 20 s to deliver 2 m l ofneurotoxin at three different levels per injection.Fig. 2. Four-cannula setup for simultaneous injection of 6-OHDA at four sites in the striatum.27513 6-OHDA Lesion Models of Parkinson’s Disease in the Rat11. Stop the pump and leave the cannula(e) in place for a further2 min, before slowly retracting from the brain.12. Flush the cannulae as above.13. Suture the skin and administer analgesia according to yourlocal SOP.14. Place animals in a heated recovery cage until fully conscious.In our laboratory, operated rats are tested for lesion efficacy using 3.3. Rotation Testingamphetamine (methamphetamine hydrochloride), at 2 weeks and 4weeks post-lesion, and using apomorphine (apomorphine hydrochlo-ride dehydrate) at 5 weeks post-lesion. Rotation is usually assessedusing an automated rotometer system, over a 90 min test session foramphetamine and a 60 min session for apomorphine. Immediatelyfollowing injection, rats are placed in 30 cm diameter circular, flatbottomed bowls, enclosed in 30–50 cm high Perspex or aluminiumcylinders. Rats are attached via a harness to a rotometer head whichsends information to a computer-controlled, automated rotometersystem (e.g. Rotomax System, AccuScan Instruments Inc.). The sys-tem records both ipsilateral and contralateral turns, from which thenet rotation (ipsilateral minus contralateral) can be calculated. Rota-tion scores are reported as either net rotations over the entire session,or as mean net rotations per minute. When an automated system isnot available, alternative methods of measuring rotation may be used.Method one is to make a video recording of each animal’s rotationsession and then count rotations from the recording using high speedplayback. A second method is to sample the rotation of each animalby recording rotation at regular intervals over the session. Animalsare observed for 1 min at a time at either 10 min or 15 min intervals,recording both ipsilateral and contralateral rotations. Scores maythen be extrapolated for the entire session.In our early studies, we used a minimum criterion for lesionsuccess as seven turns per minute under 5 mg/kg methamphet-amine. However, this dose was associated with high levels of ste-reotypic behaviour and occasional mortality, As a result, we nowuse a somewhat lower dose (2.5 mg/kg), and six net rotations perminute (540 net rotations in a 90 min session) as the criterion fora successful 6-OHDA lesion corresponding to approximately 95%depletion of striatal dopamine in studies based on post-mortemneurochemical assay.Re-lesioning of rats which do not meet this criterion is seldomsuccessful, and as a result they are usually excluded from the experi-ment. For subsequent treatments, lesioned rats are allocated intocounterbalance groups matched either on the 4-week amphetamine,or the 5-week apomorphine scores, such that prior to treatment, allgroups have approximately the same mean rotation scores.Following experimental treatments to ameliorate or reversethe effects of the 6-OHDA lesion, rotation scores are also used for276 E.M. Torres and S.B. Dunnettin vivo assessment of treatment efficacy. Pre-lesion, protective treatments, such as growth factors (e.g. GDNF) or anti-apoptotic agents (e.g. caspase inhibitors), may reduce lesion-induced dop-amine cell death in the ventral midbrain (10, 11). This is reflected in reduced levels of amphetamine rotation in the treated groups. Post-lesion treatments such as replacement of dopamine function by implantation of embryonic dopamine cells, or dopaminergic cells derived from stem cells, restore dopamine innervation to the lesioned striatum, and reduce the levels of net ipsilateral rotation. Interestingly, large dopamine grafts in the striatum can cause a net contralateral rotation (so-called over-compensatory rotation) due to their action on residual supersensitive receptors in the ipsilateral striatum (12, 13).Unilateral lesions of the dopamine system induce well character-ized deficits on the contralateral side of the body. The principal methods used to assess these deficits include the stepping, cylinder, staircase, and corridor tests, all of which detect either preferred use of the ipsilateral limbs or neglect of contralateral space. For full details of behavioural testing of unilateral lesioned animals, see the chapters by Fleming and Schallert, Smith, and Heuer, this volume and Farr and Trueman Volume II in this series.The efficacy of the 6-OHDA lesion may assess post-mortem using immunohistochemical detection of the dopamine synthesizing enzyme, tyrosine hydroxylase (TH). Successful MFB lesions induce massive loss of TH immunoreactive neurons in the ipsilateral substantia nigra compacta and ventral tegmental area. Following striatal injection of the toxin, the majority of SNc neurons are lost. Fixation, sectioning, and staining of brain tissues are beyond the scope of the current chapter. Briefly, animals are sacrificed and perfused transcardially using formaldehyde fixative (usually 4% in phosphate-buffered saline). Frozen sections may then be stained immunohistochemically using antibodies against tyrosine hydroxy-lase. For a detailed methodology, see Torres et al. (14).The rat, 6-OHDA lesion of the dopamine system is a widely used and extremely useful model for Parkinson’s disease research. The unilateral depletion of the dopamine system has little effect on the rat’s health and, after recovering from surgery, lesioned animals require no more extra care than a normal rat. Behaviourally, the loss of dopamine on one side of the brain leads to well characterized deficits, chiefly affecting use of the contralateral limbs and an attentional preference for ipsilateral space. To a greater or lesser extent, these deficits may be affected, pre- or post-lesion, by a3.4. Behavioural Assessment3.5. Post-Mortem Assessment4. Conclusions27713 6-OHDA Lesion Models of Parkinson’s Disease in the Ratrange of therapies, either by amelioration of the effect of the lesionor by replacement of dopamine in the target area.This chapter has detailed two, well tested and effective meth-ods for unilateral lesioning of the rat dopamine system. The coor-dinates used have been developed over many years and are used bya number of laboratories in the field. However, there is a consider-able variation in the coordinates used, and the results of a brief scanof papers published in 2009/2010 are shown in Table 3forreference.Table 3MFB lesion coordinates in the literature 2009/2010Coordinates (mm):reference A=L=V=Nose bara Marked depths are reported, or assumed, to be from skull surface278 E.M. Torres and S.B. DunnettAcknowledgementsOur experiments in this field are supported by grants from the UKMedical Research Council, Parkinson’s UK, and the EuropeanUnion S eventh F ramework T ransEUro, R eplaces a nd N euroStemCellprogrammes.References1. Ungerstedt U (1968) 6-Hydroxy-dopamineinduced degeneration of central monoamine neurons. Eur. J. Pharmacol. 5: 107–1102. Bjorklund A, Wiklund L, Descarries L (1981)Regeneration and plasticity of central sero-toninergic neurons: a review. J. Physiol (Paris) 77: 247–2553. 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