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Ion channels > Pain and VGSC > Flashcards

Pain and VGSC Flashcards

1
Q

Channelopathies

A
  • Human conditions resulting from changes in the function of ion channels.
  • In absense of human conditions, scientists have to take an experimental approach to determine the role of ion channels in physiological processes and predict links to human diseases (disease research).
    > Don’t have a condition per channel could be because changes in function could be immobilised by other diseases, or its a further target, or that the phenotype is subtle so its only obvious at older ages/ mixed in with aging
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2
Q

What is Pain?

A

An unpleasant sensory and emotional experience associated with actual or potential tissue damage.

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3
Q

Different kinds of pain

A

ACUTE PAIN:
Alerts body to chemical/thermal/mechanical stimuli that has potential to damage body tissue, protects the body.

PATHOLOGICAL PAIN:
- Inflammatory pains results from tissue damage and inflammatory diseases
-Neuropathic pain results from damage to nerves, which can be caused by trauma, diabetes and cancer treatment.

CHRONIC PAIN:
Pain lasting more than 12 weeks.
19% of Europeans surveyed suffer moderate to severe chronic pain
Treatment remains unmet need or over t50%.

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4
Q

Pain management - NSAIDs

A

NON STEROIDAL ANTI-INFLAMMATORY DRUGS
- Inhibition of COX enzymes which produce prostaglandins
- Limited as people who have heart and or renal conditions and those with diabetes cannot use them.
- Side effects: GI tract

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5
Q

Pain management - Opioids

A

Opioids effect receptors in CNS, PNS and GI
Short term side effects are mental confusion, drowsiness, nausea and constipation.
Can form a tolerance
Dependency -

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6
Q

Pain management - Gabapentin

A
  • An anticonvulsant (used for epilepsy) used for neuropathic/nerve pain.
    -Mild side effects in most
  • Not immediate > takes a few weeks to have an effect
  • Can be addictive if taken for a long time
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7
Q

The pain pathway

A

Can be divided by three steps carried out by three types of neurons.

  1. TRANSDUCTION
    - Conversion of physical and chemical stimuli into an action potential
    - This is carried out by sensory neurons of the Dorsal Root Ganglia.
    - PERIPHERAL TERMINAL TO CENTRAL: Skin > Primary Sensory Neurons > Somatic Sensory Pathway > Somas in DRG go to Nerve > Dorsal horn
    - Stimulus is concerted into neural signal to be transmitted to brain through specialised sensory nociceptors
  • Two ganglia per vertebrae > 31 pairs of DRG, each contains thousands of sensory neurons > these collect information from distinct spinal dermatomes
    > Innovate viscera: hands are sensitive and able to locate due to density of viscera > more diffused in other areas of body, hence harder to locate specific areas of pain.

*Peripheral terminal: Spinal ganglia goes into spinal cord and terminates in dorsal horn of spinal cord. 3 layers of membranes (meninges) protect the spinal cord (and brain) > the DRG is external of this hence it is also external to BBB

  1. TRANSMISSION
    - Transmision of the action potential from DRG neurons to neurons in the thalamus. Goes up CNS pathway.
    - Carried out by projection neurons in the dorsal horn of the spinal cord.
    - Mediated by neurotransmitted such as substance P and glutamate (binds to next neuron in pain pathway)

*Modulation of transmission: Primary synapse and the descending pathway

  1. PERCEPTION
    - Pain signal reaches the somatosensory cortex, where the intensity and nature of the signal is perceived.
    - Carried out by thalamic neurons projecting to the S1 somatosensory cortex.
    - (Emotional component of pain is processed in areas such as amygdala and anterior cingulate cortex.)
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8
Q

Modulation of Pain

A

DESCENDING PATHWAY (CNS)
*Signal reaches brain > emotional side effect can initiate descending pathways

  • Prefrontal cortex and Amygdala neurons project to Periaqueductal grey (PAG) in the midbrain.
  • PAG projects to Rostral ventral medulla (RVM)
  • RVM projects to primary synapse in dorsal horn to modulate transmission of pain signals.

*Primary afferent is linked to the secending neuron and to another interneuron to modulate the net effect of the second order neuron.
* Can release neurotransmitters such as Serotonin, Norepinephrine and Opiates from desc. and Glycine and GABA from the interneuron.

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9
Q

Pain versus Nociception

A
  • Nociception is the detection from the peripheral mechanisms (detection of noxious stimuli)
  • Central mechanisms take these signals to the brain to understand pain
  • All of this falls under “Pain”
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10
Q

DRG Neurons in Peripheral Mechanisms

A

Signal is stimulated from sensory receptor > Spinal cord > Sent to brain to understand painful nature

  • Dorsal Root Ganglia contains the somas of sensory neurons. They’re outside the BBB and are part of the PNS.
  • DRG are a heterogenous population of different types sensory neurons. Stimulated by chemical, thermal and mechanical stimuli.

Classified according to size in early discovery:
Large A beta > respond to non-noxious stimuli (touch) > Touch stimuli allows for balance and movement also
Medium A gamma and Small C > respond to noxious stimuli (pain sensing)

State of myelination of the axons that eminate from these neurons can classify >
A Beta > Thick myelin sheath
A gamma > Thin myelin
C > Naked (protected by Schwann cells but no myelination)

DRG contain transducing ion channels and receptors: Allows to transduce into an AP to send to the brain, activated by different stimuli
TRP family > Heat stimuli
P2X family > ATP activation > ATP is released by cells that have burst so indicator of damage
TRKA > immune cells release TRKA which is a growth factor
GPCRS > activated by neuromotivators.

*Mechanical stimuli is hard to find channels and ntrasm that are linked to pain

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11
Q

Role of VGSC in Pain Signalling

A
  • The influx of sodium ions into the neuron triggers an AP once a threshold is met, which travels along the sensory neuron reaching from the peripheral terminals in the skin, muscle, joints and viscera through to the DRG and eventually to the central terminals in the spinal cord to understanding in the brain.
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12
Q

Voltage- gated sodium channels (VGSC)

A
  • Essential for excitability of muscle and neurons
  • Are important for generation and propagation of action potentials
  • They are major drug targets for local anaesthesia, cardiac arrhythmias and epilepsy.
  • Mutations in VGSC can also cause inherited pain disorders such as inherited erythromelalgia and paroxysmal extreme pain disorder. Mutations can also cause hyperalgesia (increased sensitivity to pain) and allodynia (pain caused by non-painful stimuli).
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13
Q

Molecular Diversity of Mammalian Alpha Subunits

A

There are 10 cloned cDNAs which produce 9 functional VGSCs, 1.1 to 1.9 Nav

Nav1.1
-SCN1A
-CNS, PNS
-Cardiac muscle

Nav1.2
-SCN2A
-CNS, PNS

Nav1.3
-SCN3A
-CNS, PNS

Nav1.4
-SCN4A
-Skeletal muscle

Nav1.5
-SCN5A
-Cardiac muscle
-CNS, Skeletal muscle

Nav1.6
-SCN8A
-CNS, PNS
-Cardiac muscle

Nav1.7
-SCN9A
-PNS

Nav1.8
-SCN10A
-PNS

Nav1.9
-SCN11A
-PNS

(Nax > not a focus on pain at current development standpoint
-SCN7A
-heart, uterus, skeletal muscle, astrocytes, dorsal root ganglion cells)

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14
Q

VGSC as targets for Analgesic Drugs

A

Only THREE of the nine VGSC subtypes are appropriate to investigate due to expression in the PNS
- Nav 1.1, 1.2, 1.3 and 1.6 are majorly expressed in the CNS and PNS >
- Nav 1.4 and 1.5 are majorly expressed in muscle (skeletal 1.4 and cardiac 1.5)
- Nav 1.7, 1.8 and 1.9 are majorly expressed in the PNS > these are viable to be analgesic targets. Pharmacologically, VGSC Nav1.7 is the only one sensitive to TTX, 1.8 and 1.9 are resistant

*1.1 LOF leads to 10% mild epilepsy and 85% of severe epilepsy AND GOF leads to the same phenotype > important for brain function, therefore altering function comes with unwanted side effects.

  • 1.2 and 1.3 GOF also shows severe epilepsy > same isse with brain
  • Only effecting PNS eliminates worry of neurological side effects.
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15
Q

Genetic Approaches > How do you study a gene’s function?

A

MODEL SYSTEM > In vitro/in vivo
TYPE OF INTERVENTION > loss of function/ gain of function
MEAN OF INTERVENTION > Pharmacological/Genetic

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16
Q

Genetic Approaches > Classical vs Conditional Mouse Knockouts

A

CLASSICAL:
- Affects all the cells of the body in all tissues
- Knockout starts from fertilisation > initiated from removing or inactivating a specific gene in the mouse genome

CONDITIONAL:
- Directed to specific cells of interest
- Induced when needed > doesn’t necessarily start from fertilisation, allows for precise control over when and where the proteins function is lost and can help to understand its specific role in different contexts.

17
Q

Conditional KD > The Cre/LoxP system

A

CRE RECOMBINASE BINDS TO A LOXP SEQ IN DNA

  • A site is genetically engineered within the mouse to be surrounded by LoxP sites. These are targets for the Cre enzyme to bind to.
  • A seperate mouse is introduced which expresses the Cre recombinase enzyme in a specific tissue/at a specific time.
  • When these two mice are crossed, the Cre is able to bind to the LoxP sites and excise the sequence in the cells.
  • Leads to loss of function of the protein which had LoxP attached.
  • Researchers can control the tissue or time when the LoxP sites are deleted by introducing different Cre-expressing mice. This allows for the specific role of the protein to be studied in different contexts.

In Mouse 1, LoxP sites would be inserted into the Nav1.3, Nav1.7 and Nav1.9 genes.

In Mouse 2, the Cre recombinant gene is expressed in nociceptors
- Cre needs to find an endogenous gene that is expressed in nociceptors “only”. That gene has Cre DNA seq incorporated into it, and this “hybrid” gene now produces Cre enzyme in the nociceptors.
- Behavioural analysis where the heterozygous Nav1.8Cre mouse is used, shows no pain deficits. It’s important to make sure the genetic tool does not contribute to the observed phenotype.

18
Q

Tests of Pain behaviours

A

MECHANICAL PAIN THRESHOLDS: DETERMINE THE FILAMENT SIZE THAT TRIGGERS PAW WITHDRAWAL.
- Test of von Frey filaments at increasing stiffness is applied to the paw of a mouse until it reaches its threshold, withdrawing its paw. This is recorded.
- Can be used to evaluate effectiveness of pain relieving treatments/pain sensitivity > will withdraw its paw at higher thresholds.

THERMAL PAIN THRESHOLDS:
MEASURING THE TIME IT TAKES THE ANIMAL TO MOVE PAW
- Tail-flick test > Heat source is focused on the mouse’s tail, the latency for the mouse to respond by flicking its tail is measured and recorded as a threshold. Easy to perform, non invasive and the response is a clear indicator of pain. (However, also measures reflexes, sensitivity and motor function).
- Glass floor test, heat source in applied to the bottom glass floor of a cage and the time it takes for the mouse to react by jumping is measured and recorded as the threshold.
+ An alternative to the tail flick test, as glass floor does not involve physical contact to the animal and allows for a more natural response to the heat stimuli. May not be as reliable, as it relies on the mouse’s voluntary movement to jump away from the pain stimuli.

19
Q

Stimulus Independent Behaviours > Grimace Scale

A

Assesses pain based on observations of changes in facial expression that are thought to indicate pain.
- Ear position
- Cheek bulge
- Whisker change
- Nose bulge
- Orbital tightening
Scale can be used to evaluate efficacy og pain-relieveing drugs.
“Stimulus independent” > Pain is not caused by a specific event of action, but is caused from underlying conditions and diseases already prevalent.

20
Q

Stimulus Independent Behaviours > Burrowing Test

A

Ability to dig and build a nest to observe their general well-being, their motivation and their physiological state.
- Tube is filled with burrowing substrate, like sawdust or soil, and the mouse is placed at the open end of the tube, while the other end is closed.
- Behaviour of mouse is measured alongside the amount of substrate pushed out, the depth of the burrow and how complex the nest it makes is.
- Can assess use of drugs which effect mood or motivation
- Relatively simple and non-invasive, can provide measure of well-being in comparison to the norm.

21
Q

Stimulus Independent Behaviours > WBA (weight beating asymmetry) test

A

Measuring the weight bearing capacity of the mouse on each hind limb by placing the animal on a force plate and measuring the weight distribution > plate is split to a left and a right measurement.
- Used to assess pain and inflammation in mice
- Evaluate drugs/treatment on the animals ebility to bear weight on their hind limbs > cage disallows them from being on all 4
- Simple and non-invasive

22
Q

Stimulus Independent Behaviours > Gait Analysis

A

Studies how the mouse moves by using software to track and analyse its movements when walking along a long chamber. It uses cameras to record the movements.
- Evaluate drugs’ impact on ability to move and walk, which can reflect the pain and inflammation levels.
- Can also be used to assess neurological disorders > impaired function equals impaired movement
- Non invasive, can see well being

23
Q

What is Stimulus Independent Behaviour?

A

THE ACTIONS PERFORMED BY AN ANIMAL IN ABSENSE OF EXTERMAL STIMULI/CUE.
- Said to reflect the animal’s internal state, such as mood or motivation
- Grooming, burrowing, eating, sleeping > driven by animal’s internal needs.

24
Q

Pyschosocial Studies > Light/dark box

A
  • The mouse is placed in a chamber divided into two compartments, one brightly lit and the other one in darkness.
  • It can move freely between the chambers
  • The amount of time spent in each compartment indicated levels of anxiety or fear.
  • Can be modified to add other stimuli to add social interaction aspects with other mice
  • Evaluates the anxiolytic/antidepressant like effects of drugs or genetic manipulations
25
Q

Psychosocial Studies > The three chamber test

A

The study of SOCIAL BEHAVIOUR in mice
- Larger central chamber and 2 side chambers each containing a mouse or object
- The test mouse is placed in the central chamber and allowed to move freely. Depending on the time spent interacting with the objects or the stimulus mice in the chamber is used to study its social behaviour.
- Can evaluate effects of drugs or genetic manipulations that affect social behaviour. Social anxiety

26
Q

Psychosocial Studies > The elevated plus maze

A

The mouse is placed in an elevated maze in the shape of a plus. There is a central chamber which has 2 open arms and 2 enclosed arms that extend from the central chamber. It’s highly illuminated.
- Used to study anxiety-like behaviours in mice
- Time spent in the open arms as well as number of entries into the open arms are indicators of anxiety like behaviour > mice tend to avoid brightly lit, open spaces, hence a mouse that spends more time in the open arms is considered to have LESS anxiety like behaviour.
- Anxiolytic effects of drugs/genetic manipulation

27
Q

Inflammatory pain model in Mice

A
  • Inject Inflammatory agent into paw (agent can be such as carrageenan, complete freud’s adjuvant and formalin) to cause inflammation.
  • Used to study the molecular and cellular mechanisms underlying inflammatory pain
  • Can measure pain behaviour, changes in activitiy of pain-related genes and pathways, and test the effectiveness of drug therapies.
  • Have been used to study a wide range of inflamm pain conditions, including osteoarthiritis, wheumatoid arthiritis and neuropathic pain.
28
Q

Contribution of Nav1.8 to pain

A

SPECIFIC to a subset of sensory neurons > expressed almost solely in nociceptive neurons. In medium/small neurons and not so much in the larger neurons.
- Critical role in initiation and conduction of pain signals
- Associated with both acute and chronic pain
- Genetic mutations in the SCN9A gene (which encodes Nav1.8) has been linked to inherited pain disorders like erythromelalgia and paroxysmal extreme pain disorder

29
Q

How does the mix of VGSC contribute to the exciteability of a nociceptive neuron?

A
  • The neuron’s AP threshold is influenced by expressed VGSC subtypes
  • AP (VGSC) threshold is around 45mV
  • Upon stimulation, goes towards a more positive value from resting potential (~ -60mV) initiating VGSC to open
  • Gap between resting potential and threshold indicates exciteablility > the smaller the gap, the more easier it is to stimulate, hence more exciteable
  • Ion channels able to convert visual stimuli into physical membrane disturbances > brings it closer to threshold

VGSC SUBTYPES HAVE DIFFERENT GATING KINETICS
- Differs in: activation threshold value, how many ions they allow through at peak conductance, inactivation voltage
- If it can open at a more negative value, first to open and more sensitive to signals.
- More positive > open later and allow for further push to AP peak

30
Q

Knockout of Nav1.8

A

-Nav1.8 knockout: “the tetrodotoxin-resistant sodium channel SNS has a specialised function in pain pathways” 1999 > as restricted to pain neurons, shouldnt be lethal if supressed.

Tests showed it has a SMALL EFFECT on pain:
- Testing baseline response to a hot stimulus shows no change in loss of Nav1.8
- Baseline response to touch (von Frey hairs) = no change
- HOWEVER Baseline pain to strong pinch shows they were VERY resistant. No response to any pinch.

-Pathhological pain KD:
- Pain sensitivity after inducing inflamm = attenuated but not as resistant as expected
- After inducing neuropathic pain, showed no change in pain AKA still painful in the KD mouse.

Future studies:
- Leo S 2009: independent classical knockout elicited same report
- Lai J 2002: Knockdown of mRNA in adult mice using antisense oligos against 1.8 (causes Nav1.8 to be destroyed) showed further inhibition of Neuropathic pain
- Ekberg J 2006: Pharmacological block by peptide toxin (MrVIB) showed further inhibition of Neuropathic pain
- Jarvis MF 2007: Pharmacological block by synthetic blocker A-803467 (selective) attenuates neuropathic and inflammatory pain

31
Q

Difference in Nav1.8 studies

A
  1. Mouse strain variability > different mice could have different genetic coding which can affect the expression and function of Nav1.8
  2. Tissue specific expression of Nav1.8: some are also expressed in sympathetic and parasympathetic neurons, these could alter the resulting effect.
  3. Age of the mouse > expression and function of VGSC can differ when ageing
  4. Different toxins used can have different specificites, which can alter the effectiveness of their own model experiment, as they will affect different sites. This can also alter the outcome.
  5. All studies had to measure the pain in the same measurements > could be error in cases of different tests to measure could have been carried out

Nav1.8 transduction is complex, meaning that it’s difficult to predict the outcome of KD in all settings.

32
Q

Contribution of Nav1.9 to pain

A

Cloned AFTER Nav1.8 > found in nociceptive neurons (and gut neurons). Restricted to PNS and to less cells than Nav1.8

-It is thought that Nav1.9 may play a role in development and maintenance of chronic pain, as it has the ability to enhance the exciteability of nociceptors and contribute to the hyperexciteability of sensory neurons in chronic pain disorders.
- Has a greater level of expression in nociceptors hat respond to mechanical and thermal pain, and itching.

Genetic mutations in the SCN11A gene (encodes Nav1.9) which are linked to inherited pain disorders like erythermalgia.

33
Q

Nav1.8 Blockers for clinical use

A

Efficacy of Pfizer’s Pf-04531083 in treating post-surgical dental pain > trials terminated in 2012
VX-150 in phase II of trials.
DSP-2230 for neuropathic pain > phase I completed

*Doesn’t show a lot of promise

34
Q

Classical KD of Nav1.9

A
  1. Contribution of tetrodotoxin-resistant VGSC Nav1.9 to sensory transmission and nociceptive behaviour. Priest BT 2005
  2. The VGSC Nav1.9 is an effector of peripheral inflammatory pain hypersensitivity. Amaya F 2006
  3. GTP up-regulated persistent Na+ current and enhanced nociceptor excitability require Nav1.9. Ostman J 2007
  4. Exploring the role of nociceptor-specific sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice. Leo S 2009

Phenotype:
Baseline:
- Hot stimuli = no change
- Touch using von Frey hairs = no change
- Strong pinch = no change

Pain sensitivity:
- Inflammation = attenuated
- Neuropathic pain = no change

+ No Blockers for Nav1.9 has been developed

35
Q

Contribution of Nav1.3 to pain

A

Expressed in CNS and PNS. Embryonic expression of Nav1.3 fades postnatally after around 2 weeks, therefore doesnt have much room in adult morphology.
mRNA level in adult in DRG is significantly smaller than the rest of Nav channel subtypes

So why?
- Nav1.3 is UP-REGULATED in CHRONIC PAIN models. Multiple models researching into Neuropathy and Diabetic neuropathy models shows reexpression of Nav1.3 increases exciteability of pain AP > shorter breaks between AP, able to reach it sooner as threshold easily pushed to.

36
Q

Floxed Nav1.3 for Conditional Deletions

A
  • Can not be done classically as it could be lethal in embryo and devlopment vvv

Classical: Mice thrived and devlopped normally
Baseline: No change in all 3 stimuli
Pain sensitivity: No change in 2 stimuli
- Doesn’t have effect on neuropathic pain > “Sham operation” > operating and causing physical injury, does not change before and after
Wild type > injury of nerve = sensitivity increases, chronic mechanical hyper sensitivity
Classical KD > same level as WT, hence no change in pain

Conditional Nav11.3 KD STILL have no effect on Neuropathic pain
- Restriction of deletion to nociceptive Nav1.3
- KD has similar values to WT
- KD in adult/postnatal = profound hyper sensitivity, hence still no aid in pain levels (hypersensitivity)

NO NAV1.3 BLOCKERS

37
Q

Contribution of Nav1.7 in pain

A

Nav1.7 is expressed principally in sensory and sympathetic ganglia, with low expression in the CNS
- Stronger expression in pain sensing than non-pain sensing neurons

38
Q

Pain behaviour after Nav1.7 deletion in Nav1.8 positive neurons

A

Ion channels (8 decks)

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