Pain and VGSC Flashcards
Channelopathies
- 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
What is Pain?
An unpleasant sensory and emotional experience associated with actual or potential tissue damage.
Different kinds of pain
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%.
Pain management - NSAIDs
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
Pain management - Opioids
Opioids effect receptors in CNS, PNS and GI
Short term side effects are mental confusion, drowsiness, nausea and constipation.
Can form a tolerance
Dependency -
Pain management - Gabapentin
- 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
The pain pathway
Can be divided by three steps carried out by three types of neurons.
- 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
- 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
- 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.)
Modulation of Pain
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.
Pain versus Nociception
- 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”
DRG Neurons in Peripheral Mechanisms
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
Role of VGSC in Pain Signalling
- 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.
Voltage- gated sodium channels (VGSC)
- 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).
Molecular Diversity of Mammalian Alpha Subunits
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)
VGSC as targets for Analgesic Drugs
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.
Genetic Approaches > How do you study a gene’s function?
MODEL SYSTEM > In vitro/in vivo
TYPE OF INTERVENTION > loss of function/ gain of function
MEAN OF INTERVENTION > Pharmacological/Genetic
Genetic Approaches > Classical vs Conditional Mouse Knockouts
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.
Conditional KD > The Cre/LoxP system
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.
Tests of Pain behaviours
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.
Stimulus Independent Behaviours > Grimace Scale
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.
Stimulus Independent Behaviours > Burrowing Test
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.
Stimulus Independent Behaviours > WBA (weight beating asymmetry) test
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
Stimulus Independent Behaviours > Gait Analysis
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
What is Stimulus Independent Behaviour?
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.
Pyschosocial Studies > Light/dark box
- 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
