Eleven animals were rendered hemi-parkinsonian through unilateral administration of the neurotoxin 6-hydroxydopamine hydrobromide (6-OHDA, Sigma-Aldrich) via the MFB cannula to evoke unilateral degeneration of dopaminergic neurons in the nigrostriatal pathway (Tieu, 2011 (link)). As 6-OHDA also will selectively destroy noradrenergic neurons, 30 min before 6-OHDA administration animals were pre-treated with intraperitoneal injections of 5-mg/kg desipramine (Sigma-Aldrich) to protect nonadrenergic neurons and 50-mg/kg pargyline (Sigma-Aldrich) to inhibit monoamine oxidase activity (McConnell et al., 2012 (link); So et al., 2012 (link)). Anesthesia was induced using a mixture of 7% sevoflurane in 2 l min−1 O2 and maintained using 2.5–3.5% sevoflurane in 1–1.5 l min−1 O2. Animals were positioned in a Kopf stereotactic frame for intracerebral injection. Immediately before infusion, 5-mg 6-OHDA was dissolved into 2 mL 0.02% ascorbic saline (Sigma-Aldrich) stored at 4°C to produce a final concentration of 10 mM. Ten microliters of 6-OHDA solution were administered through the MFB cannula using a Hamilton syringe at a rate of 2 μl/min. Animals were given one week to recover. Animals that did not exhibit unilateral motor symptoms after the recovery period were re-infused with 6-OHDA a maximum of two additional times.
6 ohda
Manufactured by Merck Group
Sourced in United States, Germany, France, China, Spain, Sao Tome and Principe, Japan, United Kingdom, Hungary
6-OHDA is a chemical compound used as a laboratory tool in neuroscience research. It has the molecular formula C₈H₁₁NO₂. 6-OHDA is primarily employed as a neurotoxin to induce selective degeneration of dopaminergic and noradrenergic neurons in animal models, enabling the study of Parkinson's disease and related neurological disorders.
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Lab products found in correlation
Ascorbic acid, by Merck Group (42 mentions)
Desipramine, by Merck Group (26 mentions)
Apomorphine, by Merck Group (23 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (17 mentions)
L dopa, by Merck Group (9 mentions)
Stereotaxic apparatus, by Kopf Instruments (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
6 hydroxydopamine 6 ohda, by Merck Group (6 mentions)
Desipramine hydrochloride, by Merck Group (6 mentions)
Stereotaxic frame, by Stoelting (6 mentions)
Pargyline, by Merck Group (6 mentions)
Sh sy5y, by Merck Group (6 mentions)
L ascorbic acid, by Merck Group (6 mentions)
Rotenone, by Merck Group (6 mentions)
Benserazide, by Merck Group (5 mentions)
Stereotaxic frame, by Kopf Instruments (5 mentions)
5 fluoro 2 deoxyuridine fudr, by Merck Group (4 mentions)
Benserazide hydrochloride, by Merck Group (4 mentions)
Microplate reader, by Thermo Fisher Scientific (4 mentions)
Tween 80, by Merck Group (4 mentions)
353 protocols using 6 ohda
1
Unilateral 6-OHDA-Induced Parkinsonism
2
Neurotoxin Treatment of PC12 and VM DA Neurons
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For PC12 cells, 10 mM stock solutions of 6-hydroxydopamine (6-OHDA) (Tocris) or 1-methyl-4-phenylpyridinium (MPP+) (Sigma) diluted in water were freshly prepared just before each experiment. 6-OHDA was used at a final concentration of 150 μM and MPP+ was used at a final concentration of 1 mM. For VM DA neurons, the 10 mM stock solution of 6-OHDA was prepared in MEM supplemented with ascorbic acid (Sigma) to prevent 6-OHDA oxidation and degradation (Ding et al., 2004 (link)). 6-OHDA was used at a final concentration of 40 μM in 0.015% ascorbic acid. MPP+ was diluted in water and used at a final concentration of 40 μM.
3
6-OHDA Induced Parkinson's Disease Model
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The design of the experiment was similar to Voutilainen et al. (2009 (link); Fig. 1A). Stereotaxic surgery was performed under isoflurane anesthesia (4.5% during induction and 3% during maintenance) as described previously in detail (Voutilainen et al., 2009 (link)). The animals received unilateral stereotaxic injections totaling 20 µg of 6-OHDA (Sigma Chemical; calculated as free base and dissolved in ice-cold saline with 0.02% ascorbic acid, 20 µg/4 µl each) in the left striatum using coordinates relative to the bregma and dura (A/P +1, L/M +2.7, D/V –5). At the completion of each injection, the needle was kept in place for 4 min to minimize backflow of the solution. Before the 6-OHDA injections, desipramine (Sigma Chemical; 15 mg/kg, i.p., 1 ml/kg) was administered to prevent the uptake of 6-OHDA into noradrenergic nerve endings. Four weeks later, the rats were given a unilateral intrastriatal injection of CDNF (1 µg), GDNF (1 µg), CDNF (2.5 µg), GDNF (2.5 µg), CDNF (5 µg), or GDNF (5 µg) alone or combination of them (in 4 µl of PBS) into the same site.
4
Selective Depletion of Meso-Cortical Dopamine
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Rats were anesthetized with chloral hydrate (400 mg kg−1, i.p.). Thirty minutes before surgery, rats received an i.p. injection of desipramine hydrochloride (25 mg kg−1; Sigma) in order to prevent noradrenergic terminals from taking up 6-OHDA65 (link). To selectively deplete meso-cortical dopamine innervation to the M1, 8 μg of 6-OHDA (Sigma, St Louis, MO, USA) dissolved in 2 μl of sterile 0.9% saline and 0.02% ascorbic acid (or 2 μl of 0.9% saline for sham injection) was injected into bilateral M1 forelimb territory at a rate of 1 μl min−1 respectively. The micro-syringe for injection stayed in the brain for 15 min in order to prevent backflow of solution. For levodopa (L-DOPA)-treated group, 6-OHDA lesioned or sham-operated animals received intraperitoneal injection of L-DOPA (Sigma) 30 min before behaviour training. L-DOPA was given at a dose of 15 mg kg−1 dissolved in vehicle solution (saline with 0.1% ascorbic acid), combined with the peripheral decarboxylase inhibitor benserazide-HCl (Sigma) at a dose of 15 mg kg−1.
5
Unilateral Dopamine Depletion Protocol
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The unilateral DA lesion was carried out under 1-2% isoflurane (in air; Forene Abbott, Wiesbaden, Germany) general anesthesia. Additional local anesthesia (Marcain 2.5 mg/ml, AstraZeneca AB, Södertälje, Sweden), was applied in the area of the incision in the scalp, and following surgery Romefen (5 mg/kg; VET, Merial, Lyon, France) was used as post-analgesia. Briefly, in order to achieve a severe dopamine lesion in the nigrostriatal pathway, the rats were placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA), and 14μg 6-OHDA (Sigma-Aldrich AB, Stockholm, Sweden) in 4μl ascorbic acid (0.02% in saline) was injected intracerebrally at one site in the right or left MFB with the coordinate (according to the bregma): anterior-posterior: −4.4 mm; medial-lateral:±1.2 mm; dorsal-ventral: −7.8 mm below the dura. The tooth bar was set to −2.4mm for the injection. The toxin was injected into the brain at a rate of 1μl/ml, and the needle was kept in place for an additional 3 min before slowly being retracted. Thirty minutes prior to the 6-OHDA injections the selective noradrenaline reuptake inhibitor desipramine (25 mg/kg, Sigma Aldrich AB) was intraperitonally injected to protect the noradrenergic neurons from the 6-OHDA toxicity.
6
6-OHDA Unilateral Striatal Lesion
7
6-OHDA Unilateral Striatal Lesion
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Catecholaminergic neurotoxin 6-OHDA was injected unilaterally into the left dorsal striatum (AP=+1.0, ML=+2.7, DV=-4.0 mm relative to the bregma, according to the rat brain atlas [30 ] of male Wistar rats in a stereotaxic surgery under isoflurane (Vetflurane® 1000 mg/g, Virbac, France) anaesthesia [31 (link)]. Rats were fixed on a stereotaxic frame (Stoelting, USA) using ear bars and incisor bar. Lidocaine-adrenalin-solution (10 mg/ml; Orion Pharma, Finland) was injected between the skull and the scalp for local anaesthesia and to prevent bleeding. The skull was exposed, and a burr hole was made using a high-speed drill. An electronic injector (Quintessential stereotactic injector, Stoelting) and a 10 μl microsyringe (Hamilton Company, USA) were used to deliver 16 μg of 6-OHDA (Sigma-Aldrich, Germany), calculated as free base and dissolved in ice-cold saline with 0.02% ascorbic acid. Injection rate was set to 1.0 μl/min and injection volume to 4 μl. At the completion of the injection, the needle was kept in place for 4 minutes and then slowly withdrawn. Desipramine (15 mg/kg i.p.; calculated as free base; Sigma-Aldrich) was administered 30 minutes before the 6-OHDA injection to prevent the uptake of 6-OHDA into noradrenergic and serotonergic nerve terminals.
8
Neurotoxin Treatment of PC12 and VM DA Neurons
9
Optimizing 6-OHDA Induced Parkinson's Model
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6-OHDA surgery was performed 3 weeks after virus transfection. The mice were deeply anesthetized to align the bregma and lambda of the skull, and posterior holes were drilled at the specified coordinates for the localized striatum (AP: + 0.5 mm, ML: − 1.8 mm; DV: − 3 mm, − 2 mm). A solution of 6-OHDA (Sigma-Aldrich, Saint Louis, USA) with ascorbic acid saline (2 μg/μL) was then injected into the right striatum at a controlled rate of 500 nL/min, at a total volume of 4 μL. In the shamoperated group, an equivalent volume of 0.02% ascorbic acid saline was administered18 (link).
On the 7th day post 6-OHDA injection, the PD model mice were assessed using an apomorphine (APO)-induced rotation test. Following intraperitoneal injection of an APO solution (0.5 μg/g), rotations exceeding 120 within a 30-min duration were considered indicative of successful PD model establishment. Mice that passed the apomorphine-induced rotation test were randomly divided into two groups. Mice that failed the rotation test, were excluded from further experiments. The reliability of the PD model was further confirmed via immunohistochemical analysis of tyrosine hydroxylase (TH) expression in the nigrostriatal system.
On the 7th day post 6-OHDA injection, the PD model mice were assessed using an apomorphine (APO)-induced rotation test. Following intraperitoneal injection of an APO solution (0.5 μg/g), rotations exceeding 120 within a 30-min duration were considered indicative of successful PD model establishment. Mice that passed the apomorphine-induced rotation test were randomly divided into two groups. Mice that failed the rotation test, were excluded from further experiments. The reliability of the PD model was further confirmed via immunohistochemical analysis of tyrosine hydroxylase (TH) expression in the nigrostriatal system.
10
Unilateral Parkinson's Rat Model
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The method to cause unilateral SNc damage was the same as described previously (Fan et al., 2011). In brief, the rats were anesthetized with 4% chloral hydrate (400 mg/kg, intraperitoneally) and pretreated with desipramine (Sigma-Aldrich, St. Louis, MO, USA), dissolved in normal saline, 25 mg/kg, intraperitoneally, to protect norepinephrine neurons from 6-OHDA toxicity. Subsequently, the rats were fixed on a stereotaxic frame (SN-2N, Narishige, Tokyo, Japan). 6-OHDA (Sigma-Aldrich; dissolved in saline containing 0.01% ascorbic acid, 8 µg/4 µL) was injected into the right SNc (anteroposterior – 5.0–5.2 mm, lateral 1.9–2.0 mm, dorsal 7.1–7.2 mm; Paxinos and Watson, 2005). The injection was made at a rate of 0.5 µL/min using a glass micropipette connected to a 10 µL microsyringe. At 1 week after the operation, the rats were injected subcutaneously with apomorphine 0.05 mg/kg body weight (the apomorphine, Sigma-Aldrich, was dissolved in saline containing 0.01% ascorbic acid). Only rats that rotated to the unlesioned side more than 20 times every 5 minutes were selected for the subsequent experiment.
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