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1
Q

protocols of whole brain examinations (common investigations)

A

Whole brain is fixed in formalin 2-3 weeks before examination.
The hindbrain is often seperated with the cerrbellum being removed.
The top of the brain is commonly investigated to look for signs of atrophy indicated by wider sulci.
The bottom of the brain is often investigated to looked for inflamation and squashing of the brain.
Saggital sectons are used to investigate te ventircualr system.
Thin progressive cuts are used for indepth analysis using staining procedures such as immunohichemistry (anitbodies) and staining to assess cellualr pathology and morphology .
Staining examples are the H+E stain, Nissl stain

2
Q

What is the H+E stain?

A

This is the Haemotoxylin and Eosin stain

3
Q

what can we look for using immunohistochemistry or other staining procedures?

A

Immunohistochemistry allows the digitaton of findings and the assessment of cell density of stained cells.

imunohistochemistry specificity can allow for multiple staining of specific cells and leayers of the cortex.

Immunohitsochemtisry can be used to ientify swollen of achromatic neurons, signs of inlfamation and other neurodgeenrative conditions.

Can be used to identify sepecific cells and thus abnormal cells that act as biomarkers of disease for exampe HIRONO cells in AD.

Other stains can be used to look at the anatomy of cells. For example identifiying Eosinophillic neurons (lacking neuronal detail).

4
Q

describe a method of identifying amyloid protein

A

Amyloid protein form a beta sheet confrirmation. this structure alters its optical propeties in reponse to polarised light (BIREFRINGENCE).

staining with Congo red and exposing smaples to polarised loght shows a APPLE-GREEN BIREFRINGENCE.

5
Q

important neurological landmarks in the investigation of astrocytes in disease?

A

Immunohostochemistry can be used to identify them by trageting GLIAL FIBRILLIARY ACID PROTEIN (GFAP).

in Injury astrocytes become larger as a result of hypertrophy, hence this can be used as a biomarker of disease.

astrocytes can be found in Glial scars following CNS lesions.

Alot of tumours ae gliomas these stem from dividing astrocytes. GFAP cytology can be used to assess the extent of Gliosis.

6
Q

How can we apply immunohistochemisry to the assessment of oligodendrocytes?

A

Oligodendrocyte are produced from rogenetior and immunohistochemistry can be used to assess the maturity of glia.

Given their importance to CNS myelination and a detecton of less glia or less mature glia could be a sign of demyelination which is seen in codton such as mutliple sclerosis.

7
Q

possible insights from marking for microglia and macrophages?

A

Microglia are important in the inflamatory resposne, responding to damge or infection through enetering a ramfied state.

for example. in inflamtory disease such as encephalitis the microglia will surround neurons.

Macrophges surrounding blood vessels, froming perivascular plaques.

8
Q

what is subcortical laminar heteropia and what is the normal Doublecortin (DCX) function? This is an example of cortical displasia.

A

This is the formation of a second band of neurons under the cortical plate.

This stems from defficient neuronal mriration during developemnt and a cause of leanring dissabilties in later life.

Cortical displasia is common in cases like epilepsy also.

This can be attributed to mutations on the X-chromosome in a gene called DCX, a microtuble associated protein (MAP) that palys a key role in neuronal migration.

9
Q

How can we look for inflamation.

A

MRI- inflamtion reslts in the release of extravascualr fluid that has a hyperintesity on the image (white blots in MS)

Staining for microglia and Macrophages

  • Macrophages will suround vascualrtue forming perivascular plaques
  • Microglia in response to insultu enter an inflamed state, or Ramifed state. There long processes and hypertrophy appearance show this.

Neurons.

  • Lookig at the morphology f neruons they can appear inflamed or achromatic.
  • we can assess morphology using haemotoxilin and Eosin stains.

hypertrophy of atrocytes.

squashed base of the brain

Look for markers. E.G cytokine identification.
- Look for their mRNA,
- Wetsern blot to qunatify abundance of protein.
-

10
Q

Describe the pros and cons of IPSCs and mouse models in the study of neurodegenerative diseases?

A

MOUSE MODELs
+ve / they are extremely genetically felexible allowing for indepth investigation of the imoacts of genetics discovered in humans.

+ve They are extremely easy to analyse behaviourally with several established trials to test apects f memory, motor incoordination e.t.c.

+ve they have relatively high breeding rates and short life spans and so there is a ready supply and you can investigate them postmortem at several different stages of lif, cannot do this with a human brain. (INVIVO)

-VE Although their life spans are accelerated relative to ourthis is not so on a linear time scale. this means that in the case of neurodegenrative diseases that progress over life and with age being the biggest risk factor for many they present very late in life these trodents simply may not live long enough to accumulate a toxic threshold of dysfuncton. possil leading to false negatives (this appears to hav been the case in many mice studies of disrupted mtiophagy.)

INDUCED PLEURIPOTENT STEM CELLS (IPSCs)

+ve these can be produced directly from the fibroblasts of patients and thus allowing for the investigation of a mutation in the context of its genetic environment.

+ve can be used to induce a wide range of different neurons. excellent fo cases like PD when a specific SUBSET is degenrated. (Chung et al 2016 used methods to produce midbrain DA neurons that expressed comparale levels of DA neurons markers and had the same calcium meadited tonic pacekeeping aactivity of SNpc neurons.

  • ve cannot produce all forms of neurons
  • ve takes a long time for neurons to mature, its is difficult to investigate late onset diseases as we currently cannot aritifcially age neurons.
  • ve only can be studies invitro
  • ve difficult to investigate function within the context of a neural circuit. although organoids can be produced they are not perfect replicas of the real thing. Hence the neural architecture of neruons is often not maintained.
11
Q

list two different mitochondrial dysfunctions and briefly explain how they contribute to neurodgenerative disease?

A

.MRC dysfunction. This results from oxidative stress and ROS production and mtDNA mutations. this are seen in PD and HD

PTP- activation can drive calcium flooding and activation of the apoptosis pathway. resulting in cell death.

12
Q

what is the difference between primary,secondary and tertiary prevention

A

Primary- this attempts to prevent the onset of disease

Secondary- This attempts to trrea the undelrying pathology and cure disease

Tertiary- This attempts to treat the symptoms of the disease.

13
Q

EXPLAIN THE POSSIBLE MECHANISMS BY WHICH REPEAT NUCELOTIDE EXPANSIONS CAUSE NEUROPATHOLOGY AND GIVE THREE EXAMPLES OF NEURODEGENERATIVE DISEASES.

A

Essentially LOF, Dipeptide repeat, toxic gain function, RNA toxicity

Hexanucleotide repeat GGGGCC- ALS and FTD. RNA can be translated in sense and anti-sense direction C9orF72- 3 mechs of toxicity

  • LOF th natural function is unkown but losing this could be toxic. the role has been pottentailly link to being a GEF fro small RAb GTpases giving it a function key to lysomal biogensis and autophagy.
  • RNA toxicty- this may alter RNA production as a reuslt of the large repeat sequence producing an abberant RNA traps RNA in nucleus which. The Repeats form loopd called quadruplexs that allow for atypical pairing, this allows for the binding and trapping of RNA-binding proteins in the nucleus. dirupting their function in RNA processing. RNA foci formation and the sequestration of proteins away from where they are needed, have been observed in ALS tissue
  • Protein toxicity- the long repeat can cause an abberant processing of RNA called REPEAT ASSOCIATED NON-ATG translation (RAN-translation). This forms toxic DIPEPTIDE REPEAT PROTEINS, which are aggregation prone and interfere with RNA processing.

Support from study in FTD studies in drosophilla. Thos soley expressing GGGGCC repeat expanded RNA (no protein) where innefected. suggeststing dipeptide repeats mediates excitotoxicity.

GAA-Repeats Friedreich ataxia- autosomal reccesive ataxia stemming from deffiencient Frataxin levels as a result of GAA repeat expansion. a protein found in mitochondria and key to function in particular iron chelation to prevent promotion of ROS poduction. henc disruption allows for increased oxidative sress and mitochondrial dysfucntion. (less effiecient energy production.)

CAG- repeats- HTT and SCA- HTT is key to abbernat splicing and production of toxic protein fragments. misfolding and toxic gain of functionin ataxin inSCAs.

14
Q

List 4 common pathogenic mechanisms contributing to various neuroddgenerative diseases.

A

Mitochondrial dysfunction.- Seen across almost all neurodegenrative disease we can see disruptions such as caclium buffering capacity, membrane pottential and reduction in MRC and ETC activity (big in PD (complex-1, mitoophagy, also MND, oxidative stress in all.)

Excitotoxicity- this is related to glutamate metabolism alteration or other mechs resutling the the calcium flooding of cells. Linked to huntingdons heavily with decreased glutamate uptake and increase NMDA recptors presentation on MSN in HD)

Protein aggregation- A commonality across many is both cytoplasmic (NFTs(AD), LBs (PD), stress granules (MND and Ataxia) and intranuclear inclusions (HTT (HD), and extracellular aggregations (AB plaques in AD).

Synaptic dysfunction- Linked to many. recently linked with the AB solublke oligomers in AD.