Resultados para la clasificación de fallas usando extracción de

For all in vivo experiments, stereotaxic surgery was carried out by Miss D. Pearce and Dr. S. Harper and rats perfused by Dr. M. Rigby. Sections were processed and embedded in paraffin wax by Mrs. A. Jennings. All cutting, immunostaining and quantification was carried out by myself.

2.4.1. 6-OHDA lesions and perfusions

Medial forebrain bundle lesion experiments were carried out in accordance with the Home Office Animals (Scientific Procedures) act 1986. Animals were housed in a pathogen free environment and maintained on a 1 2h :1 2h light dark cycle with

the minimum required to demonstrate reliable effects. Surgery was carried out under aseptic conditions. Following surgery the animals were placed in a heated incubator until fully awake. All animals were checked 4 times daily in the first 48h and then daily thereafter, by trained animal care staff. Body weights were recorded in order to monitor animals’ welfare.

In these studies male Sprague Dawley rats in the weight range of 250-300g were used. Animals were anaesthetised with isoflurane until deep anaesthesia was obtained as determined by loss of paw withdrawal and blink reflexes. Animals were shaved over the top of the head and the skin was swabbed with antiseptic solution. Animals were placed in a David Kopf stereotaxic frame. A cut was made in the skin and bregma was exposed by scraping the surface of the skull. Once the location of bregma was determined a small hole was drilled in the skull at a position -2 . 2 in the anterior-posterior direction and -2 .0mm in the medial-

lateral direction. Once the depth of the dura was determined the canula was lowered to a position -7.9m m below dura. 8 pg of 6-hydroxydopamine/ascorbate

solution (2.5mg/ml reconstituted with sterile PBS). Cannula was lowered into position, left for 1 minute, then 60H D A was infused at Ip l per minute (3.2 min) and cannula was left in place for a further three minutes to prevent diffusion back up the tract left by the cannula.

After the required amount of recovery time for each experiment (48 hours to 14 days), animals were deeply anaesthetised with Euthatal (1 ml/kg) until there was no paw withdrawal reflex. Animals were perfused with a solution of 4%

formaldehyde in saline, and brains were processed for paraffin embedding. 6pm

sections were cut throughout the substantia nigra, and two placed on each slide. Typically, every slide was immunostained throughout the substantia nigra.

2.4.2. Quantification of TH-immunoreactive neurones in in vivo models

To quantify the cell loss following unilateral 6 -OHDA lesion, sections were

immunostained using a polyclonal primary antiserum raised against TH. Sections were dewaxed and rehydrated through graded ethanol. Sections were dewaxed in two changes of two minutes each in xylene, then rehydrated through two changes of 100% ethanol, one change of 95% ethanol and one change of 70% ethanol, each at two minutes per treatment. Sections were placed in running dH2 0 for two

minutes, then washed 3x2 minutes in PBS. Endogenous peroxidase was blocked by incubating the sections in PBS/0.3% H2O2 for 30 minutes at room temperature.

Slides were washed 3 times with PBS, and immunostaining carried out.

For immunostaining, the Biogenex Optimax automated slide staining system was used. A one day immunostaining protocol was used. All reagents were prepared in proprietary Optimax buffer, and all steps were carried out at room temperature. Prior to immunostaining, a pap pen was used to draw across the top and bottom of each slide above and below the sections. Non-specific binding was blocked by 30 minute incubation in 5% normal goat serum; blocking buffer was then removed without washing and rabbit polyclonal anti-TH was added at 1:1000 in 5% normal goat serum. Sections were incubated in primary antibody for two hours, then were washed twice and secondary antibody added. The secondary antibody used

was goat anti-rabbit IgG, biotin conjugate, prepared from the Vectastain Elite ABC kit by adding one drop of antibody to 10ml 5% normal goat serum (approximately 2.5pg/ml final antibody concentration). Sections were incubated in secondary antibody for 30 minutes; following this treatment, slides were washed and incubated in peroxidase conjugated avidin-biotin complex, prepared from the Vectastain Elite ABC kit as the manufacturer’s instructions one hour prior to addition. Slides were incubated in ABC reagent for 30 minutes, then were washed and peroxidase visualised using diaminobenzidine (DAB) prepared from the Biogenex DAB kit. Slides were incubated in DAB for 10 minutes, then were washed once with dH2 0 , and three times with PBS. Nuclei were

counterstained using haematoxylin, which was fixed using acid alcohol (0.5% HCl in 70% ethanol) prepared immediately prior to use. Following counterstaining, sections were dehydrated and cleared following the reverse of the dehydrating and dewaxing protocol previously described, and slides mounted with coverslips using DPX.

For quantification of TH-immunoreactive cells in the substantia nigra and the VTA, slides from all animals within a study were pooled, randomised and blinded by another investigator. Sections were observed on a Leitz DMRB microscope. Counts were made of the TH-immunoreactive neurones in the nigra and VTA of both the ipsilateral and contralateral hemispheres. Once all sections were quantified, slides were unblinded. Data were presented in the form of ipsilateral counts versus contralateral counts to allow observation of uniformity of lesion throughout the nigra.

2.4.3. Immunostaining for phosphorylated c-jun

Expression of phosphorylated c-jun was visualised using a rabbit polyclonal antibody raised against serine 63 phosphorylated c-jun. For staining of phosphorylated c-jun, cultures were dewaxed and rehydrated as described above. Antigen retrieval was carried out; sections were immersed in lOmM citrate buffer (pH 6) and heated on full power in a microwave for two bursts of five minutes,

with the buffer being topped up in between. The slides were then allowed to cool in the citrate buffer for 5 minutes and then washed with PBS. In the first experiments, comparisons were carried out of sections stained with and without antigen retrieval to allow comparison. Immunostaining was carried out as described above, using a primary antibody dilution of 1:100. All secondary antibody, ABC reagent and DAB development steps were as described above.

To colocalise phosphorylated c-jun expression with TH immunoreactivity, double immunolabelling was carried out. Phosphorylated c-jun and TH are expressed in different cellular compartments, with TH being expressed in the cytoplasm and phosphorylated c-jun being expressed in the nucleus; it was thus possible to carry out double peroxidase labelling using DAB and Vector SG. For this technique, cultures were first immunostained using the phosphorylated c-jun primary antibody at 1 : 1 0 0 dilution; this was detected using a biotinylated secondary

antibody, followed by peroxidase conjugated avidin-biotin complex and the staining visualised using Vector SG. Vector SG staining produced intense black staining of phosphorylated c-jun immunoreactive nuclei. Slides were washed, and peroxidase quenched by I hour incubation in 0.3% H2O2 prepared in PBS. Slides

were then blocked using 5% normal goat serum, and immunostained for TH using the rabbit polyclonal TH antibody. This primary antibody was added at a higher dilution, 1:5000 compared to 1:1000 previously used; the staining was thus fainter, and allowed clearer visualisation of double immunolabelled cells. Following primary antibody, biotinylated secondary antibody was added, followed by peroxidase conjugated avidin-biotin complex and DAB. The TH immunoreactivity was thus visualised as light brown cytoplasmic staining, with the phosphorylated c-jun staining appearing as dark black nuclei. Sections were haematoxylin counterstained, dehydrated and mounted using DPX as described above.

To quantify P-jun immunoreactive cells, and double labelled TH/P-jun labelled cells, slides were blinded by another investigator. Labelled cells were counted in both ipsi- and contralateral nigra and VTA; only cells with a visible nucleus were counted. In double labelling experiments, the total number of TH cells and the number of TH/P-jun double labelled cells were counted in both the nigra and VTA in the ipsilateral and contralateral hemispheres. After all quantification was complete, slides were unblinded and the data analysed.