Methods

Question 1

What is the purpose of chapter 2 methods?

Answer 1

Two major things:
1. To provide a detailed description of the methodology that is present across all chapters of the thesis.
2. To provide a roadmap and guide for the methodology of all chapters.

Question 2

Why did you choose Methoxy-x04? Why not other options?

Answer 2

Methoxy-x04 is one of the oly commercially available and established probe for in vivo use for passing the BBB.

Other approaches fail to show BBB permeability, are not a suitable wavelength or have not been proven for in vivo use.

Currently, it is the most suitable option.

Question 3

What are the other options compared to Methoxy?

Answer 3

Curcumin - been used for retinal staining of plaques
Thioflavin T - used for histology
Congo red - used for histology cant cross BBB
CRANAD - new approach but need infrared imaging
Akulumine - luciferase based amyloid reporter

Question 4

What do the following terms mean and what do Methoxys values mean:
- MW
- pKa
- logPoct
- Ki?

Answer 4

MW - size of the molecule –> want it low to cross the BBB

pKa - how acidic/alkaline –> want it neutral

logPoct - the phase between octneals and acques - lipophoilivity - want it high

Ki - the inhibition constatn - binding efficiency to the target –> want it low as it shows it can disrupt other bidning

Question 5

What colour of wavelength is Methoxys excitation and emission?

Answer 5

purple
blue

Question 6

Why did I use that concentration of Methoxy-x04 and in that diluting solution?

Answer 6

I did a literature search of papers that have used Methoxy-x04 and extracted the dosing information.

I found that the majority of papers, including the landmark study use a drug concentration of 5 mg/ml in that solution.

Question 7

Did I test other concentrations? Why not?

Answer 7

No

Because this was well established and previously used.

Also, with concentrations in vitro I got positive signals.

In conclusion, testing a higher concentration may provide a stronger signal, however this would saturate binding sites quicker.

Question 8

How did you choose the in vitro concentrations?

Answer 8

I began with a range of concentrations and upon testing I found the range that provided the best signals.

By this, I mean signals that were not completely saturated at highest inputs and provided a signal at the lowest input.

Question 9

You mentioned difficulties with the Methoxy-x04 being soluble. Why?

Answer 9

The suppliers note that Methoxy-x04 is not soluble

Question 10

Did you try other methods of injection?

Answer 10

The original paper note that i.v. injection can supply strong signals marking the plaques at a quicker rate.

We did a pilot of a couple of animals attempting this in an acute condition.

Eventually we decided against this route of administration as for freely behaving this is stressful, you cannot be certain the full dose is being administered and repeat dosing on the tail vein could be problematic.

Question 11

Did the mice have any behavioural or physiological changes after the dose? Have any toxic effects of Methoxy-x04 been recorded?

Answer 11

Without quantifying the behaviour, we found no changes in behaviour or weight of the mice. We also had no fatalities or health conditions in mice with repeat injections.

Additionally, a paper that tests various small molecules including Methoxy tracked body weight and health and showed no changes compared to the control group.

Question 12

How did you follow the 3Rs?

Answer 12

Reduce: by completing repeat recordings on the same mice, I reduced the requirement of several different groups of mice which reduced the n number greatly.

Refine: I refined the urethane dosing strategy to improve survival for a full recording. Also, I modified the head-fixation of mice to reduce the stress.

Replace: completed mock pharmacokinetic and calibration experiments using stock solutions in order to gather information requried for establishing the system without using mice.

Question 13

What does congenic mean?

Answer 13

An inbred strain of mouse that contains a small genetic region (ideally a single gene) from another strain, but which is otherwise identical to the original inbred strain.

Question 14

What does >F10 mean?

Answer 14

This is because the mice used were purchased from Jackson Labs.

For several years in the lab they have been breeded with C57 to continue the litter.

This has resulted in an inbred litter that has been bred more than 10 generations.

Question 15

How do you confirm the genotype of mice?

Answer 15

The litter will produce both positive and negative littermates.

They will be ear tagged and the labelled tissue will be shipped to an external supplier that will complete using real-time PCR by Transnetyx.

This will amplify the DNA to determine if the FAD mutations are present or not.

Question 16

Were any randomisation or blinding completed? If not, why?

Answer 16

For chapters 1 and 2 no blinding was completed as due to breeding conditions, the genotype was known before use. However, there was randomisation in the start times for PK and RD experiments across mice with some starting morning and others in afternoon. However, once the recording was started, the same time was maintained across days.

For chapters 3 there was randomisation in the treatment protocol by establishing a timetable before experiments were completed. Therefore, the groups of mice that were assigned to either AV or NS for their last treatment was blinded.

Question 17

What were the mice used for training?

Answer 17

This includes cadavers that were used when I was first learning how to complete specific surgical procedures including terminal anaesthetic, craniotomies, recovery anaesthetic and head-cap surgery.

Some of these were recovery procedures and allowed optimisation of implantation approaches.

Question 18

What was the survival rate of these procedures?

Answer 18

100%

In terminal procedures, due to the instability of urethane, sometimes mice died before the full recording. However, this was rectified as much as possible by refining the dosing protocol.

All mice survived recovery procedures. Occasionally, after several months use the fibre would become unstable and mice were perfused.

Question 19

Why did you want to align images to the atlas?

Answer 19

It was vital for me to confirm that the fibre had passed the same brain regions and to allow quantification of plaques in the same brain regions on the hemisphere that has not been damaged from the fibre implant.

Question 20

How is the plaque detection automated?

Is it successful?

Answer 20

The software goes through all sections and determines any pixels that are over a specific size and intensity and then marks this as a cell.

By optimising the parameters this allows good detection of cells that can be manually accessed.

Question 21

Why did you use AMaSiNe and not another software?

Answer 21

When starting analysis, I considered various options.

The most established approach was Sharptrack which is often used by high profile groups for identification of the neuropixels probe. However, this requires manual alignment and cell detection using an extra ImageJ plug in.

Therefore, we adopted for amasine as this allows automated everything and they provided all codes to allow for modification for our specific requriements.

Question 22

In Fig 2.4B, why is one of the images more faint than the others?

Answer 22

This is because there may be some dust or something that is altering the auto brightness adjustment on the microscope camera. However, when fully stitched this is restored.

Question 23

What does yaw and pitch mean for alignement?

Answer 23

Yaw and pitch are angles of the hsitological seciton that they estimate.,

Rotation around the side-to-side axis is called pitch. Rotation around the vertical axis is called yaw.

Question 24

Why is the fibre annotation not just one single line?

Answer 24

Sometimes depending on the alignment of the skull in the stereotaxic frame, the fibre could be implanted at a slight angle.

The same can occur when slicing the brain for histology.

Also the fibre is 200 um thick so will be across several sections.

Therefore, there is dmage that can be spread across various sections and depending on the deformation and alignment angles, they may be slightly warped. This is why we form a regression model of these points to estimate the fibre track.

Question 25

Why did you quantify plaques at 200 um?

Answer 25

This was an arbituary value as it is difficult to determine the distance the light of the fibre will dissipate due to scattering within the brain.

However, some papers have tried to quantify this and this has resulted in the value of <500 um.

Question 26

Why did you quantify the contralateral hemisphere? Is plaque pathology consistent?

Answer 26

As the fibre is implanted into the brain tissue, this causes some tissue damage and therefore loss of plaque pathology at the recorded region.

Therefore, to get a better representative we quantify the other hemisphere. As the sections are aligned, this will be the same brain regions.

However, we made an assumption that plaque pathology was consistent across brain regions. Recent studies disprove this. We done some analysis on the contralateral hemisphere and found those with damage can interfere with accurate measure.

Question 27

How are the images binarised?

Answer 27

Using a built in MATLAB function they determine a blob feature on the image of a certain size and threshold. This removes the background of this identified feature.

Question 28

How do they align the non-anchor images?

Answer 28

Equal distances between the AP axis

Question 29

Why did you choose those parameters for plaque detection?

Answer 29

By manual assessment following automated detection, these parameters provided the best detection by having the fewest false positives/negatives.

If the detection was bad, these parameters were changed which occured often with FAD- models as this could pick up several noise as a plaque.

Question 30

Why did you choose GENUS as the treatment to investigate?

Answer 30

GENUS has recieved alot of attraction due to the papers finding a dramatic reduction of plaque pathology, with others suggesting the opposite.

As this is a non-invasive approach, it is apptractive option, cheap and easy to implement.

Also, no study investigates the real-time effects which may help uncover the differing findings suggesting this approach.