Mechanisms and mediators

Question 1

Why are murine models used so extensively in neurovascular research?

Answer 1

We want a good model to replicate things that are happening in humans in terms of conserved mechanisms - if it doesn’t it wouldn’t be a good model

Animal models are good because in combination with fMRI we can use other high resolution technologies to investigate mechanisms and mediators in greater detail

1. Pathways are very similar to the human system - same topographic system
2. A complete body map on the surface of the cortex can be seen using histology
3. Rat or mouse somatosensory cortex is located at the brains surface making it accessible to a range of technologies including 2D-OIS

Question 2

What makes a good model of disease?

Answer 2

Properties of a good model:
1. Well known anatomy
2. Easy to stimulate
3. Region accessible for easy measurement

Question 3

What are the pros and cons of anaesthetised imaging?

Answer 3

PROS
High degree of control
Can be more invasive
A good anaesthetic can mimic the quiet awake state

CONS
The animal isn’t awake - hard to compare to human fMRI studies
The anaesthetic itself can lead to an alteration in neurovascular function

Question 4

What are the pros and cons of awake imaging?

Answer 4

PROS
The animal is awake - can ask more behaviourally relevant questions
More comparable to human fMRI

CONS
The animal is awake - behaviour can confound your results
You need to measure behaviour - more equipment needed
More time consuming - all animals need training - welfare implications

Question 5

What are the advantages of ex-vivo slice research?

Answer 5

Allows amazing spatial precision, isolate single cells, capillaries
High throughput pharmacology experiments can be performed
These are the only experiments that have successfully shown the role of pericytes

Question 6

What are the disadvantages of ex-vivo research?

Answer 6

Lack of blood flow - how can you perform neurovascular research when there is a non-functioning vascular system
Timescale for responses usually over minutes - too slow and potentially non-physiological

Question 7

What are the key cells in the neurovascular unit?

Answer 7

Astrocytes
Pericytes
Endothelial cells

Question 8

Why have astrocytes long been thought to be the controller of neurovascular function?

Answer 8

They are in the ideal position to communicate signals from the neurons to the vasculature
Astrocyte endfeet surround all blood vessels in the brain

Question 9

What did Zonta et al. (2003) find?

Answer 9

Measured calcium responses in neurons, endfeet of astrocytes and the diameter of arterioles
Found that increased Ca2+ evoked in the astrocyte processes, through the activation of mGluRs, was found to propagate to the perivascular endfeet
The timing of this propagation was correlated with the dilation of the arterioles that were in direct contact with the astrocytic endfeet
Direct electrical stimulation of an astrocyte also caused an increase in vessel diameter
This strongly implicates the role of astrocytes in vasodilation

Question 10

What is the prostaglandin pathway?

Answer 10

Excitatory neuron releases glutamate
Glutamate binds to receptors on astrocytes
This causes an increase in calcium which activates the phospolipase A2 pathway
This activates arachadonic acid and COX 1 and COX 2 enzymes which causes prostanoids to be released
Prostanoids are known to dilate blood vessels

Question 11

What did Zonta et al. (2003) find regarding COX inhibitors?

Answer 11

Used pharmacology in both slice and in vivo they could inhibit the dilation response
CBF measurements before and after intravenous injection of mGluR receptor antagonists
Over 50% of the dilation response gone

Question 12

What did Takano et al. (2006) find?

Answer 12

In vivo anaesthetised adult mice
2-photon microscopy
Photolysis of caged Ca2+
Showed similar results to Zonta - COX inhibitors produced a 50% reduction in blood flow
Again suggested it was COX that mediated the response

Question 13

What is the problem with the findings from these papers researching astrocytes?

Answer 13

Inhibition of the COX pathway only reduced the blood flow response by 50%
There could be a redundancy in the system - another system could be stepping in when the COX pathway is removed

Question 14

What are pericytes?

Answer 14

Cells that wrap around capillaries and are known to have contractile proteins associated with them

Question 15

What did Peppiatt et al. (2006) find about pericytes?

Answer 15

Ex vivo slice experiments in either the retina or cerebellum
Paper mostly shows that when a pericyte is activated it causes capillary constriction
Nice effect but not really related to the positive BOLD response - we need dilation
However, when glutamate was administered it showed dilation but only where the capillary was ensheathed with the pericyte - could suggest their role in NVC

Question 16

What did Hall et al find?

Answer 16

When you make an electrical stimulation, the capillary dilates first and then the penetrating arteriole dilates after by actively relaxing pericytes - responses too slow, anaesthetised mouse

Question 17

What are the limitations of Hall et al?

Answer 17

The responses are far too slow - 4sec lag between capillary bed and penetrating arteriole
Anaesthetised animal

Question 18

What other contradicting evidence is there for the role of pericytes?

Answer 18

Hill et al (2015) - Regional blood flow in the normal and ischemic brain is controlled by arteriole smooth muscle cell contractility and not by capillary pericytes
Also recorded responses from awake animals - however responses still look a little strange

Question 19

What is the overall summary of pericytes?

Answer 19

Like astrocytes they are in the correct place
However, in vivo evidence is lacking showing that pericytes doing anything active to cause the dilation of blood vessels
Still very controversial with strong arguments even in terms of defining what a pericyte is
They may have a really important role in disease

Question 20

What is the role of nitric oxide?

Answer 20

Well known to be a modulator of baseline vascular tone in the peripheral microcirculation
NO is released as a freely diffusible gas and exerts its main affects on vascular smooth muscle cells

Question 21

How is NO produced?

Answer 21

By an enzyme in cells called nitric oxide synthase
Exists in several iso-forms:
- iNOS - inducible - only thought to be involved in pathology
- eNOS - present in endothelial cells
- nNOS - present in cortical neurons - mainly interneurons

Question 22

What are the mechanisms of NO?

Answer 22

In an endothelial cell
eNOS acts on L-arginine to produce L-citrulline and creates NO as a byproduct

In nerve cell
nNOS acts on L-arginine to produce L-citrulline and creates NO as a byproduct

In smooth muscle cell
NO taken up by smooth muscle cell
Activates soluble guanylate cyclase
Converts GTP into cGMP which decreases Ca2+ and causes relaxation of the cell

Question 23

What have experiments done to test the effects of NO?

Answer 23

Inhibiting NOS which stops the release of NO

Question 24

What inhibitors are used to block NO?

Answer 24

L-NNA, L-NAME inhibit both eNOS and nNOS
7-NI - inhibits just nNOS

Question 25

What did Lindauer et al. (1999) find?

Answer 25

Measured blood flow responses to whisker stimulation with LDF
Inhibition of both eNOS and nNOS with L-NNA
Conclusion that NO from neuronal NOS is a permissive factor in neurovascular coupling because the response can be re-established with an NO donor

Question 26

What did Lauritzen’s lab find?

Answer 26

Focussed on the cerebellum
Suggests NO is the dominant mediator in this region of the brain

Question 27

What is the overall summary of NO?

Answer 27

Really important modulator of baseline vascular tone
No conclusive evidence it is the main mediator of coupling - except in the cerebellum

Question 28

What is the role of the endothelium in NVC?

Answer 28

Iadecola and Berg et al showed that once the capillaries had picked up the dilate signal, a wave of backward upstream dilation propagated up the vessel wall through the endothelium
Probably via gap junctions between all the smooth muscle cells

Question 29

What did Hillman et al. find regarding the role of endothelial cells?

Answer 29

Showed the backwards dilation that other labs have shown
Used light to disrupt the vascular endothelium and stopped the backwards dilation
Wide-field disruption strongly suggests there are separate mechanisms for the capillaries and surface vessels

Question 30

What are caveolae?

Answer 30

Small invaginations on the endothelium
Can breed mice to have no caveolae and these mice have disrupted NVC
Paper suggested that caveolae in CNS arterioles mediate NVC

Question 31

What did the paper find about caveolae?

Answer 31

When whiskers of transgenic mice are stimulated without caveolae the haemodynamic responses are 50% reduced without affecting neuronal activity
Used awake animals

Question 32

What was a limitation of this caveolae study?

Answer 32

Cav mice have really bad lungs – if you put a normal mouse in a pool it will swim around for 40 mins and then sink but cav mice sink straight away. This means the cav mice didn’t move at all on the ball which could be a reason for the results – need to measure locomotion in cav mice and wildtype mice

Question 33

What are the conclusions from endothelial work?

Answer 33

Might be the most important of the neurovascular mechanisms
It translates the initial small response at the capillary bed and delivers it to the large resistance vessels
The role of the endothelium might change in disease
Caveolae might be a really important component of the response
Overall cells and mediators of the NVU appear to give the brain some redundancy - if one fails another mechanism steps in