Anatomy and neurochemistry of the substantia nigra in the wild-type sheep brain

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Chapter 3. Methods

Animals

The development of the sheep model of HD is described previously (Jacobsen et al., 2010). Briefly, pronuclei of fertilised eggs from South Australian Merino sheep were microinjected with full-length human HTT cDNA containing 73 CAG repeats and implanted into surrogate ewes. Six of the subsequent lambs were found to be transgenic, and therefore became the founders of the six lines of HD transgenic sheep. The most well characterised line to date, and the line used for the present studies, is the OVT73 line, originating from animal G0/5 (Jacobsen et al., 2010).
The sheep are housed at the South Australian Research and Development Institute (SARDI) in accordance with the SARDI/PIRSA Animal Ethics Committee (approval number 19/02). Animals were kept in large paddocks typical of the South Australian farming environment, in mixed flocks of control and transgenic animals, with rams and ewes housed in separate flocks.
For the present study, samples from 3 cohorts were used; 6 months of age, 5 years of age, and a larger 5 year old cohort of rams (Table 3.1). The cohort of 6-month-old sheep contained 7 wild-type and 7 transgenic animals, which were harvested in September 2010. The cohort of 5-year-old animals contained 6 wild-type and 6 transgenic animals, which were harvested in June 2012. The ages of these animals ranged from 64- to 70-months, and contained both rams and ewes. The final cohort of animals were harvested in August 2014, and contained 12 wild-type and 13 transgenic animals. The ages of these animals ranged from 68- to 69-months and contained only rams. For the purposes of this thesis, the 5-year-old cohort harvested in 2012 will herein be referred to as 5 year mixed cohort, while the 5-year-old cohort harvested in 2014 will be referred to as 5-year ram cohort.

Tissue Collection

All tissue collection was performed at the South Australian Research and Development Institute (SARDI) or the South Australian Health and Medical Research Institute (SAHMRI) in Adelaide, South Australia. Animals were euthanised by intravenous injection of Pentobarbitone Sodium solution (Lethabarb 1ml per 2kg bodyweight), followed by rapid exsanguination. The brain was extracted from the skull intact, then placed into a custom-made cutting matrix in ice-cold phosphate-buffered saline (PBS) to allow accurate coronal cuts to be made. The brain was blocked into 6 regional coronal sections, and divided into two hemispheres (Figure 3.1). Coronal blocks from the left hemisphere were rapidly frozen for fresh tissue analysis, while right hemisphere blocks were immersion fixed for 48 hours in freshly prepared 10% buffered (0.1M phosphate buffer pH 7.4) formalin at 4°C, then transferred to 0.1M phosphate buffer plus 0.1% sodium azide for shipment to Auckland, New Zealand. Fixed blocks were cryopreserved in 0.1M phosphate buffer plus 20% sucrose for one week. The blocks were then transferred to 0.1M phosphate buffer plus 30% sucrose for a further week. At the end of the seven days the blocks were removed from the sucrose solution and frozen with dry ice snow, and stored at -80°C until use.

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Tissue processing
Tissue block sectioning and sampling methods

The first region of interest for this thesis was the substantia nigra (SN), located on block 1 of the frozen sheep tissue. The SN is a small lens shaped nucleus, found in the midbrain of the sheep. The second region of interest was the caudate nucleus of the striatum, located on block 5 of the frozen sheep tissue. The caudate nucleus is a deep forebrain structure made up of a head, body and tail, which lie lateral to the lateral ventricles.
Blocks of tissue containing the region of interest were fixed onto a freezing microtome (Microm HM450) using OCT tissue plus ® compound (Scigen). Sections of tissue 50µm thick were precisely cut and stored in 48-well plates in phosphate buffered saline (PBS) plus 0.1% sodium azide at 4°C until further use.
For stereological analysis, tissue sections were sampled in an unbiased serial manner. Every 15th section was taken from a randomly determined start point between 1 and 15 sections from the start of the region of interest. For densitometry 3 sections per case (rostral, middle, caudal) were selected. For characterization of morphological and neurochemical composition of brain regions in control sheep, 1 section from the middle of the region of interest was selected from 3 representative control cases.

Immunohistochemistry

Immunoperoxidase labelling

Free floating sections were initially transferred to 6-well plates and washed overnight in PBST (PBS with 0.2% triton X-100) at 4°C, then incubated in a solution of 50% methanol and 1% H2O2 for 20 mins at room temperature (RT) to block endogenous peroxidase activity and prevent non-specific background staining. Standard washing of free floating sections consisted of three 10 minute washes in PBST (3 x 10 mins in PBST) unless otherwise specified. Following the methanol incubation sections were washed 3 x 10 mins in PBST then incubated with the primary antibody (Table 3.2) diluted in immunobuffer (1% normal goat serum (NGS) in PBS with 0.04% merthiolate plus 0.2% Triton-X) for 72 hours at 4°C. Optimal primary antibody dilutions were determined through preliminary studies involving serial dilutions (Table 3.3). For information on antibody specificity see appendix I. Following primary antibody incubation sections were washed 3 x 10 mins in PBST then incubated in the appropriate biotinylated secondary antibody (see Table 3.3) diluted in immunobuffer for 24 hours atA RT. Subsequently sections were washed 3 x 10 mins in PBST and incubated in tertiary antibody streptavidin peroxidase (ExtrAvidin, Sigma, dilution 1:1000) diluted in immunobuffer for 4 hours at RT, before being washed again 3 x 10 mins in PBST. For visualisation, sections were incubated in 0.05% diaminobenzidine tetra hydrochloride (DAB, Sigma) in phosphate buffer with the addition of 0.01% H2O2 to initiate the tertiary complex reaction. The optimum incubation period for the DAB solution was determined through optimisation studies to obtain the appropriate incubation time required to produce a clear brown reaction product. Free floating sections were then thoroughly washed 3 x 10 mins in PBST before being mounted onto slides with gelatin and left to air dry. Once dry, sections were rinsed in distilled water and subsequently dehydrated through a graded ethanol series to xylene (5 mins each water, 75%, 85%, 95% ethanol, 10 mins each 100% ethanol (x2), 20 mins each xylene (x2)). Each slide was removed from xylene and immediately coverslipped with DPX mounting medium (BDH Laboratory Supplies). All sections from control and HD sheep from the same cohort were processed in the same immunohistochemical batch to decrease any variability in staining.

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Immunofluorescent labelling

sheep brain. The initial steps of the immunofluorescent protocol were carried out as described for immunoperoxidase labelling. Briefly, sections to be labelled were incubated in PBST overnight at 4°C, then washed 1 x 10 mins in 1x PBS. Primary antibodies (Table 3.4) were applied collectively, diluted in immunobuffer, and incubated for 72 hours at 4°C. Following primary antibody incubation, sections were washed 3 x 10 mins in 1x PBS then incubated in the appropriate fluorescently labelled secondary antibodies (Table 3.4) diluted in immunobuffer overnight at room temperature.

Chapter 1. Introduction 
Chapter 2. Literature Review 
2.1 Huntington’s Disease
2.2 Basal ganglia function and dysfunction in HD
2.3 Animal models of HD
2.4 Gene silencing therapeutics for HD
2.5 Aims of this thesis.
Chapter 3. Methods 
3.1 Animals
3.2 Tissue Collection
3.3 Tissue processing
3.4 Imaging and Analysis
3.5 Statistics
Chapter 4.  
4.1 Introduction
4.2 Methods
4.3 Gross anatomy and cell morphology of the sheep substantia nigra
4.4 Neurochemical expression patterns in the sheep substantia nigra
4.5 Discussion
Chapter 5. Substantia nigra cell number and volume in the transgenic sheep model of Huntington’s disease
5.1Introduction
5.2 Methods
5.3 The substantia nigra pars compacta in the Huntington’s disease transgenic sheep
5.4 The substantia nigra pars reticulata in the Huntington’s disease transgenic sheep
5.5 The substantia nigra pars lateralis in the Huntington’s disease transgenic sheep
5.6. Densitometric analysis of neurochemical markers in the substantia nigra of the Huntington’s disease transgenic sheep
5.7. Discussion
Chapter 6. The caudate nucleus in the transgenic sheep model of Huntington’s disease 
6.1 Introduction
6.2 Methods
6.3 Characterisation of the striosome and matrix compartments in the wild-type sheep brain
6.4 Volume of the striosome and matrix compartments of the caudate nucleus in the Huntington’s disease transgenic sheep at 6 months of age
6.5 Volume of the striosome and matrix compartments of the caudate nucleus in the Huntington’s disease transgenic sheep at 5 years of age
6.6 Discussion
Chapter 7. RNA interference trials in transgenic sheep 
7.1 Introduction
7.2 Methods
7.3 Density of GABAAα1 receptor subunit in the basal ganglia after RNAi treatment
7.4 Density of substance P in the basal ganglia after RNAi treatment205
7.5 Discussion
Chapter 8. General Discussion 
8.1 Introduction
8.2 Summary and implications of findings
8.3 Technical considerations
8.4 Future directions
8.5 Final remarks
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Characterisation of the anatomy and neurochemistry of the basal ganglia in a transgenic sheep model of Huntington’s disease

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