Mechanisms And Mediators Of NVC Flashcards
Why are murine models are used so extensively in neurovascular research?
Want a good model to replicate things happening in humans in terms of conserved mechanisms.
Animal models are good as in combination with fMRI we can use other high resolution technologies to investigate mechanisms and mediators in greater detail - do multimodal methods to see the responses we need to understand the mechanisms of NVC.
Properties of a good model:
Well known anatomy
Easy to stimulate
Region is accessible for easy measurement
BOLD fMRI can measure responses in different regions
Even when got a BOLD signals at different regions of the brain, it doesn’t mean NVC is the same in each structure as the mechanisms aren’t necessarily heterogeneous
Strengths of anaesthetised animals
High degree of control
Can be more invasive
A good anaesthetic can mimic the quiet awake state
Quick
Limitations of anaesthetised animals
Animal isn’t awake so hard to compare to human fMRI studies
Anaesthetised can lead to an alteration in neurovascular function - need one that doesn’t impair coupling between neural activity and blood flow
Strengths of awake animals
Can ask more behaviourally relevant questions
More comparable to human fMRI
Limitations of awake animals
Behaviour may confound results if the animal restraints or fails to attend
Need to measure the behaviour so more equipment is needed
More time consuming - animals need to have surgery, then recover, then be trained - more effort and time for less animals
Advantages of ex-vivo slice research
Has led to some major advances
Allows great spatial precision, isolation of single cells and capillaries
Can use the slice for multiple experiments by washing it off = more productive
These are the only experiments which have successfully shown the role of pericytes
Disadvantages of ex-vivo slice research
Lack of blood flow - how can you perform neurovascular research when there’s a non-functioning vascular system
Time scale for responses is usually slower - doesn’t mean they’re invalid
The health of the slice - temperature plays a big role so some studies warm it up to body temperature for more reliable results (makes slice decay faster though)
Key cells of neurovascular unit
Astrocytes - form glia of brain, end feet at synaptic cleft and on blood vessels - ideal location to communicate signals from neurons to vasculature
Pericytes - has contractile proteins in to open or close capillaries so could drive NVC; ideal location
Endothelial cells
Astrocyte research - Zonta et al 2003
Slice ex-vivo
Electrode stimulated neurons
Measured calcium increase in astrocytes and the diameter of nearby vessels
Found when stimulating the neuron, astrocytic calcium increases
Direct electrical stimulation of the astrocyte caused an increase in blood vessel diameter = important for NVC
Experiments to see if you can inhibit the dilation response
Slice work:
Use COX inhibitors = stop release of prostanoids so stop NVC = good evidence prostaglandin pathway controls NVC
In vivo work:
CBF measurements before and after mGLUR antagonists and found over 50% of the dilation response is gone - redundancy in the system so can’t just be astrocytes
Takano et al 2006
Used 2 photon calcium imaging to replicate Zonta et al (2003) results with better spatial and temporal resolution
Suggested calcium could be main increase driving NVC and generating a BOLD signal
Problem with astrocytic calcium findings
The calcium response was occurring after vessel dilated but if astrocytes were the main driver surely it would happen before? Points to other mechanisms being involved
Pericytes
In capillary bed known to wrap around capillaries and have contractile proteins associated with them so could be important for baseline blood flow cos at ideal location
Peppiatt et al (2006)
Ex-vivo slice work
Stimulating pericytes caused contraction of capillary bed related to negative BOLD signal
When added noradrenaline to the slice as a pre-constrictor and when added glutamate it dilated the vessel
Suggested pericytes could do both construction and dilation
Summary of pericytes
In vivo evidence is lacking to show pericytes doing anything active to cause blood vessel dilation - no calcium change evidence or other activity change to suggest this
Thought they are involved in baseline blood flow change
May have a very important role in disease
Role of NOS in NVS
Released from neurons and vessels and causes dilation
Not inhibited by BBB - moves freely
Known modulator of baseline vascular tone
Exists in several isoforms:
- iNOS = only thought to be involved in pathology
- eNOS = present in endothelial cells next to blood flow
- nNOS = present in cortical neurons, mainly interneurons
All these act in same way to produce NOS
Lindauer et al (1999) - NOS as a modulator of NVC
Measured blood flow responses to whisker stimulation with LDF
Remove NOS and the blood flow response to whisker stimulation decreases (not 0) but when added noradrenaline it back in artificially or recovers = NOS needs to be present as a modulator bur not as a mediator cos taking it out simply dampens the response not gets rid of
Offenhauser - NOS in cerebellum
Suggested blood flow response goes down dramatically when inhibit NOS in cerebellum so suggests NOS is the dominant mediator of NVC in the cerebellum - blood flow response is driven by nNOS interneurons releasing NOS so NVC is controlled by NOS in cerebellum - mediators aren’t necessarily the same in all brain regions
Offenhauser - NOS in cerebellum
Suggested blood flow response goes down dramatically when inhibit NOS in cerebellum so suggests NOS is the dominant mediator of NVC in the cerebellum - blood flow response is driven by nNOS interneurons releasing NOS so NVC is controlled by NOS in cerebellum - mediators aren’t necessarily the same in all brain regions
Summary on NOS
V important modulator of baseline vascular tone
No conclusive evidence it’s the main mediator of coupling apart from in the cerebellum
Backwards dilation
Used light to disrupt the vascular endothelium and stopped the backwards dilation - blocks dilation completely so proof of principle backwards dilation is very important in getting blood flow response to the brain
Non-caveolae mice
When stimulating whiskers of non-caveolae mice the haemodynamic responses were significantly reduced without affecting neuronal activity
When these were put in water they drowned but normal mice swum around = don’t exhibit locomotion and have bad lungs
Summary of endothelial work
Might be the most important of the neurovascular mechanisms
Translates the small response at the capillary bed and delivers it to large resistance vessels
Its role might change in disease but need further experiments to quantify this
The caveolae might be very important components of the response
Cells and mediators of the NVU appear to give the brain some redundancy - if one bit fails something else steps in to allow blood flow to be controlled to some level
