Stroke Flashcards
Discuss evidence for the role of pericytes in capillary diametre regulation
Pericytes are found attached to capillaries and work via to regulate their dilation via different mechas to those in the smooth muscle of the arterioles.
Studies have shown that the products of neuronal activity linked via astocytes can vary cappillary diamtre via action of pericytes.
- the down stream effects of glutamate action at the post-synaptic neurons can be related to the NOS driven production of NO which only drove the dilation of arteriole smooth muscle.
- Cappilary dilation was modulated by varying effects, ATP release acts at astrocytes on ATP activated ion chanel P2X1, this drives caclium entry which activates phospholipase D2 and this acts on phosphlipids to produce phosphatidic acid and eventually Diacyglycerol. DAG is broken down by DAG lipase to eventaully produce arachidonic acid. AA is a traget fro the action of cyclooxidase 1 in the production of prostogalandins. The production of PGE2 here works to acts on EP4 (GS-CAMP-PKA-k+ phos and effluX) receptors of the pericytes to drive dilation via the relaxation of pericytes.
Hall et al 2014
- Investigation in rat cortical slices.
-Labelled pericytes with Ds RED under the NG2 pomorter. They electrically stimulated the whisker pad of mice in the somatosensory cortex whilst imaging their vasculature. They reported increased dilation of vasculature. The cappilaries dilated first. Investigating the net change in vasculature showed that cappilary dilation was repsonsible for 84% of the change.
-This faster timecourse is consisitenet with a faster time coure of action and they showed that the diametre changes where larger at the site of pericytes. Indicates they are actively releaxed.
- They also found Blocking for Pge2 action of EP4 had a inhbitory effect on the dilatory effects of glutamate much larger than other blocks. This would appear to be the driver and is consistent with the above pathway.
Hall et al 2014 reported the recording of K+ efflux in pathc clamp recordings following glutamate release in cortical brain slices.
- Investigating the effects f ischaemia they used OGD and block of ATP synthesisi to show this. They found that this resulted in a sustained decline in cappilary diamter that coorrelated with the constrcition of cpapilaries which was followed by progressive increase in pericyte Death.
- To discpover the cause of this they investigated this using AMPA (NBQX) and NMDA (AP5) and showed they cpould half pericyte death suggesting this was excitiotoxic.
- This suggests that pericytes die in rigor and thus explains sustained pericyte death. This offers a pottential target to stop prolonged hypoxic damge in ischaemia.
This could explain studies that showed that india ink capillaries are not reperfused in brain slices following middle cerrebellar aretery occlusion.
Recording in both with patch pipetes on pericytes.
- Peppiat et al 2006- Retinal slices-Studies stimulating pericytes found that they caused site specific reductions in cappilary diametre, constricted them.
*this could be related to the constricition of pericytes folllowing calcium ion entry shown by inward flow calcium currents. (stimulation only induced constiction if calcium present (removal of extracelllar calcium prevented stimulation indued contraction.) - cerrebellar slices. they also show Further support for the importance of info that Locus coerulus NA postive neuerons. 65% of those terminating on vasculature terminate on capillaries - tlikely acting on pericytes. mimickin LC NA release caused constriction on pericytre location in vasculature (glutaamte cause dilation)
marked location using NG2 proteoglycan marking. - has also shown the communication between pericytes via the endothelium on capillaries shown the conduction of the pottential. The showed that stimulating one pericyte could cause the temporal matched vasodilation of the sites of distal pericytes.
This has been attributedto gap junctions between the pericytes and the endothelium. (stimulation of 1 showed propagation currents in pericytes downstream)
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pericyte constiction and death in ischaemia has now been linked to the long lasting constriction and lack of perfusion through capilaries.
what is the prevalence of stroke?
Stroke occurs in 1 in 6 female sand 1 in 5 males.
It equates to one eery 2 seconds world wide and every 5 minutes in the UK.
This results in both mental and motor impairment.
Why is it important to treat stroke ptients as quickly as possible?
The brain is highly energy demanding, using around 20% of the bodies energy.
Anoxia is stroke confers a lack of oxygen and this is also seen with a a loss of glucose. The bulk of energy is produced via oxidative phosphorylation, and thus this is disrupted and energy production ceases causing cell death.
Every hour later the onset of stroke millions of neurons and billions of synapses are lost and it is predicted the individual ages an avergae of 3.6 years.
What are the 2 main types of stroke
Ishaemic stroke:
- This is the result of an occlusive clot that leads to a loss of blood flow don stream. 87% pf clots.
- This usually stems from 2 situations:
- Atrial fibilliation- the abnormal contraction of the heart which can lead to the formation of a clot which can move down stream
- Fat deposition- this is related to the formation of atheromateous plaques which are notorious for having pieces break off if occuring in a major artery this could travel to key cerebellar arteries causing occlusion.
Haemorhagic stroke-
- This is the results of the brusting of a blood vessel and the flooding of blood into the cranium. This is commomly a result of head injury.
- this can be intra cranial or subarachnoid. both often lead to death.
Outline the main vasuclar circuitry of the brain
The complex nerual vasuclature is designed to limit the effects of occlusion and is known as the Circle of willis.
see diagram
- The primary artery is the basialr artery, this feeds into the cricle of willis.
-at its base at the posterior cerebellary arteries, these are tasked with feeding the visula regions in the calcarine sulcus and regions of the fusiform gyrus.
- the posterior cereballr arteries are connected to the mddle cerebellar ateries and the internal carotid arteries via the posterior communicating arteries. The middle cerrbellar artery can be seen to bifurcate to feeed both the basal ganglia and regions like the brocas around the the lateral sulcus.
- The middle cerrebellar arteries are connected to the naterior cerenellar arteries via the anterior communicating arteries. the anterior cerebellar arteries feed to motor cortex.
This major arteries are part of 2 key ciruits. the cortical system which consists of vessel penertrating through greay matter into white matter and those feeding the grey matter.
The ganglionic system is made of 4 branches and is associated with feeding the thalami and the striatum.
These are termed terminal arteries. They have limited anatsomatic branches and thus feed limitted areas.
Outline some of the pottential treatments of Ischaemia?
TPF- tissue plasminogen factor is used to break down occlusive plaques.
issues - requires a scan to confirm that the stroke is cause by an occlusive clot and needs to be administered within 4 hours of clot onset to be effective.
Catheta- This is an invasive non chemical method which involves the xray guidance of inserted filament with a insera shelter device at te end to the clot. there it can clasp and remove the clot.
this only works for large vessels.
Expalin why Age is a risk factor in Ischaemia
With AGE issues like atrial fibirllation, high blood pressure become more common. many eve need blood thinners.
The higher risk of clot confers a greater risk of stroke as 87% of stroke is caused by oluccive clots.
What is the issue with imaging to assess for the treatment of clots? What study showed this and what aea of research did this prompt?
Imaging is commonly used to show that loss of perfusion in arteries and the restoration of perfusion post-treatment,
However, this can only image large arteries and there is growing evidence that these smaller vessel, in particular capillaries are nor reperfused.
A study injected india to visualise neuronal vasclature and simulate blood flow. They found mimicking stroke by occluding the middle cerrebellar artery caused a loss of perfusion which was restored by removing the block. However, The bulk of Capillary vasculature was not restored.
Hall et al 2014
In depth analysis stimualting the whisker of mice found that the cappilaries dilated before the aterioles and closer analysis showed that the changes in cerebral blood flow were dominatly mediated y cappilary dilation (84%) so they are key. (We can now link this rapis change to the neruovasculature link between neruonal activity and pericyte action at cappilaries via astrocytes.)
This prompted research into what causes this lack of restoration and led them to pericytes which regulate cappilary diametre.
What arer the roles of pericytes
Pericytes have several roles.
- maintain the BBB
- regulate the entry of immune cells
- aid glial scar fromation in the spine
- and importantly tey regulate cappilary diametre
Outline a study showing the importance of capillary action to mediation of cerebral blood flow.
A study (hall et al 2014) stimulated the whiskers of mice whilst measuring the imact on vasclar dilation. They found that the cappilaries dilated before atrioles identifiying a hierachy of vasculature. Further analysis was able to attribute the CBF shift to specific vascualture and they attrributed around 84% to cappilary dilation.
This led them to beleive that this was an active process. Looking at the sites of pericytes using DsRED (For NG2 proteglycan promoter) labelled pericytes shows that they dilation at these sites was greater.
What are the role of pericytes in capillary actions
Perictytes exist bound to cappilaries and undergo Calcium ion mediated constiction via its role in the myosin-head- actin cross bridge cycling. This in tern reduces cappilary diametre. (ATP is key here fro relaxation and myosin head detachment)
This has been shown in studies by peppiat et al 2006) showing direct stimualtion of pericytes reduced cappilary diametre but only in the presence of calcium.
- they also indentified the progation of pottenial down the endothelium and between pericytes. this inter pericyte commnication occurs via gap junctions between themselves and the neothelium allowing for collaborative constriction.
There action has now been shown to be regulated by the activity of neurons. and linkage of neruonal activity via astrocytes. Stimulation of neurons in yound adult mice caused capillary dilation.
- Cappilary dilation was modulated by varying effects, ATP release acts at astrocytes on ATP activated ion chanel P2X1, this drives caclium entry which activates phospholipase D2 and this acts on phosphlipids to produce phosphatidic acid and eventually Diacyglycerol. DAG is broken down by DAG lipase to eventaully produce arachidonic acid. AA is a traget fro the action of cyclooxidase 1 in the production of prostogalandins. The production of PGE2 here works to acts on EP4 receptors of the pericytes to drive dilation via the relaxation of pericytes.
Hall et at 2014 showed blocking of PGE2 action at EP4 stopped glutamate induced action.
Further support comes from imaging of Locus coerulus NA postive neuerons. 65% of those terminating on vasculature terminate on cappilaries. and act on pericytes.
mimivckin LC NA release caused constriction on pericytre location in vasculature peppiat et al 2006
marked location using NG2 proteoglycan marking.
How do neurons and astrocytes regulate pericyte activity?
There action has now been shown to be regulated by the activity of neurons. and linkage of neruonal activity via astrocytes.
Studies in young rats found neuronal stimualtion evoked constriction.
- peppiat et al 2006 marking pericytes using via theirexpression of NG2 proteoglycan.
- Mimicking glutamate release induced dialtion of capilaries at pericyte locations
- mimicking NA release showed constriction at these location.
- Cappilary dilation was modulated by varying effects, ATP release acts at astrocytes on ATP activated ion chanel P2X1, this drives caclium entry which activates phospholipase D2 and this acts on phosphlipids to produce phosphatidic acid and eventually Diacyglycerol. DAG is broken down by DAG lipase to eventaully produce arachidonic acid. AA is a traget fro the action of cyclooxidase 1 in the production of prostogalandins. The production of PGE2 here works to acts on EP4 receptors of the pericytes to drive dilation via the relaxation of pericytes.
Further support comes from imaging of Locus coerulus NA postive neurons. 65% of those terminating on vasculature terminate on cappilaries. and act on pericytes.
How can ischaemia be related to pericyte dysfunction?
Ischaemia has been shown to induces the constricition of Perciytes, this has been releated to increased intracellular calcium do to deficits in the ATP pump activity in anoxia. This was demonstarted in MIDDLE CEREBELLAR ARTERY OCCLUSION (MACO) mice a model of ischaemia. (in these models endothelial cells are not killed)
The reversal of pericyte constriction relies on the action of ATP to release myosin head from cross-bridge links. tha Anoxia occuring in iscahemia prevents oxidative phophoryaltion and thus adequate ATP procuction preventing this happening.
Hall et al 2014
- Investigation in rat cortical slices.
-Labelled pericytes with Ds RED under the NG2 pomorter. They electrically stimulated the whisker pad of mice in the somatosensory cortex whilst imaging their vasculature. They reported increased dilation of vasculature. The cappilaries dilated first. Investigating the net change in vasculature showed that cappilary dilation was repsonsible for 84% of the change.
-This faster timecourse is consisitenet with a faster time coure of action and they showed that the diametre changes where larger at the site of pericytes. Indicates they are actively releaxed.
- They also found Blocking for Pge2 action of EP4 had a inhbitory effect on the dilatory effects of glutamate much larger than other blocks. This would appear to be the driver and is consistent with the above pathway.
- Investigating the effects f ischaemia they used OGD and block of ATP synthesisi to show this. They found that this resulted in a sustained decline in cappilary diamter that coorrelated with the constrcition of cpapilaries which was followed by progressive increase in pericyte Death.
Hall et al 2014 reported the recording of K+ efflux in pathc clamp recordings following glutamate release in cortical brain slices.
- To discpover the cause of this they investigated this using AMPA (NBQX) and NMDA (AP5) and showed they cpould half pericyte death suggesting this was excitiotoxic.
- This suggests that pericytes die in rigor and thus explains sustained pericyte death. This offers a pottential target to stop prolonged hypoxic damge in ischaemia.
Hence, given the key role of cappilaries to CBF this may explain the long-lasting deficits in CBF observed in iscahemia.
The death of pericytes has been related to excitotoxicity as providing Calcium block or glutamte block prior to reoxygenation can reduce death, they same is said for no reoxygenation. This was carried out in oxygen and glucose derpivation (OGD) models.
Other methods like nimodipine which slow the constriction of pericytes was able t reduce death. This would slow the onset of hypoxia in the region.
The mainained vascular occlusion could then be linked to maintained anoxia and neruonal death via anoxia and so targetting perictytes to prevent maintained consticition in ischamia could be a possible therpaeutic target..
How does glutamate kill neurons in stroke?
Glutamate abundance is extrememly well regulate due to its toxicity to cells. extracellular levels are usually maintained to around 2-micro molar but in ischaemia spikes in extracellular glutamate are observed upto toxic levels ofver 100-200 micromolar.
- This is linked to anoxia and the subsequent deficits in ATP.
- Glutamate is usually taken up by astrocytes and converted to glutamain by Glutamine synthase.
- Uptake occurs va a co trasnport with Na+, K+,H+. witin this 3 NA, 1 H, 1 Glutamate goes in and 1 K GOES OUT.
- This is disrupted in iscahamae due t inbalance of ion gradients neccesary here. regualtors like the 3na-2k pump require ATP and thus stop working. this is responsible for the increased and decrease k and Na extracellular respsectively. Thisconfers a loss of fucntion in the vital role of astrocytes to buffer K plus and Glutamate to stop signalling and keep them brief.
*The subsequent spikes in K+ and spikes in intracelular Na can worsen the situation by facilitating the reversed transport throuh the glutamate transporter increasing glutamate further.
- ROSSI ET Al 2000- using v-clamp to assess the neuronal activity of CA1 neurons which are vulnerable in the ischameic hippocampus. They used a solution to mimic ischaemic conditions and observed a growing inwards current that reached a peak, that they termed the ANOXIC depolarisation. Blocking glutamate activity greatly reduced this showing it was largely a result of glutamate activity. applying AP5 (NMDA) and NBQX (others) sequentially showed this was largely the NMDA channels.
- after further study they preloaded neuronal slices with a glutamate analogue (PDC) that was transported alot slower than normal gluatamate. When then investigated the anoxic depolarisation and glutamate induced current seen in ischaemia they found they were significantly reduced. This could then be attributed to the inhibition of reversed uptake of glutamate by the the selctive binding of PDC to trasnporters and preventions of increased glutamate via this mechanim. Thus the concluded this was a key mechanism by which glutamate levels are peaked in ischaemia.
These toxic levels of glutamate will act on NMDAR to fasciliatate the flooding of cells with high doses of caclium ions whcih can cause dgeenration in seeral ways.
- osmotic bursting. The rapid entry of anions into the cell can reduce the water pottential prompting water entry via osmosis ans triggering popping of cells.
- This same efect can be mediated by action of acid sensing ion channels. The same dysfucntion transporters stopping glutamate uptake stop H+ uptake increasing there levels and thus activating ASICs fasciliatate the entry of Na and CA further into neurons down the conc grad. not only can this contribiute to osmotic popping
H+ release can contirbute to acidosis and activation of PH activated proteases and DNA endonucleases. (lactate release following increase glycolysis)
- Calicum activated proteases- Calcium entry can go on to activate damaging caclium activated proteases, like calpain. This has been shown to lyse the NCX (Na/CA transporter, thus further prolonging calcium dysregulation.
- caclium overloading of mitochondria- a key caliu regualtion is its storgae in mitochondira achoeved by the calcum uniporter. over load in this circumstance can cause the depolarisation of mitchondria leading to dysfunction and prevention of any possible ATP production and offering targetting of mitochondria for death via mitophagy. This can cause malfunction whcih is linke dto the production of free radicals ROS, are involved in pathogenic oxidation of lipases, protein and DNA damage.
- mitochondrial verload can aslo activate the releas of cytochorme C which goes on to activate the capase cascade resulting in apoptosis. (Caspase 6 activated- Caspase 3 activation which actovates DNase which goes and causes fragementation of DNA.
Thi sis demonstrated by OUYANG 1999
- Exposing rats to an iscahemia poaradigm resulted in alterations in mitochondrial morphology.
- Immunostaining for cytochrome C showed a shift from dominatly mitchondiral expression to now more diffuse cytoplasmic epxression. (This has also been shown in previous stduies showing that MCAO model rats, when unihemis[pheric, had increased DNA fragmentation and immunohistochemistyr of Cyt C in cytosol that was not seen on the non ischemic side. in fact western blot 0showed this side to have more mitochondrial cyt C.)
- Ouyang al showed Wetsern blot confirmed increased levels of phosphoryalted AKT- This is associated with alteration of transcirption in combat with apoptosis.
- The concommitant activation o detah and survival pathways may explain the delayed neruonal detah in ishaemic stroke.
NMDA activation is also linked to the activation of neuronal Nitric oxide synthase. although this is usually related to the production of NO for the relaxation of smooth muscl in vasculature here it can also contirbute to free radical formation. forms toxic peroxynitirte and competes with 02 at complex IV.
how does the brain attempt to reduce the effects of glutamate early in ischaemia (counter excitiotoxicity)
Early on the hypoxia will induce the breakdown of extracelullar ATP and AMP to produce adenosine through th activation of membrane transporters like ectonucleodases.
Adenosine is then released to act on presynatpic neurons to attenuate the release of glutamate.
They also act of presynaptic adrenosine receptors to inactivate NMDARs.
