Methods in pain research 3

Question 1

what are the problems associated with defining pain ?

Answer 1

it can be hard to separate stimulus from the response
subjectivity of the pain sensation - the same degree of pain can cause different suffering between individuals and in different situations even when the amount of action potentials produced is the same

Question 2

what are the problems associated with the use of animal models in pain research ?

Answer 2

• it is necessary to use animal models to study pain however we cannot be certain that the behavioural responses of the lab animals is related to the pain
• its difficult to grade the pain present in rodents into knowledge about humans - this is a big problem because when treatments are taken from animal models to clincial trials 99.9% of them fail because they eother dont work or produce side effects

Question 3

what are the ethical and legal issues associated with pain research ?

Answer 3

anthropocentric morale vs animal rights - its cruel

pain vs suffering- try to minimise suffering as much as possible

Question 4

what is the behavioural test for mechanical hyperalgesia ?

Answer 4

von frey hair aesthiometer

• application of filaments increasing in diameter to determine pain thresholds
• as the diameter of the hairs increases the mechanical force icreases

Question 5

what behavioural tests can be used for thermal hyperalgesia ?

Answer 5

animal is placed in a cage with a transparent floor
- a device which is basically a light will increase its temperature and it also has an infared sensor that detects the positioning of the paw - when the animal starts to feel pain it will move awa

Question 6

what behavioural tests are used for spontaneous pain ?

Answer 6

injection of inflammatory mediators into the paw of the animal - then you can look at how much the animal spends attending to its paw such as licking it

Question 7

what mediators are used in animal models of inflammatory pain ?

Answer 7

carrageenan 
complete freunds adjuvant (CFA) 
bradykinin 
prostaglandines
can use different ones to look at acute vs chronic inflammation

Question 8

what is the McGill pain questionnaire?

Answer 8

it is a list of groups describing different feelings of pain

• it is used by pain physicians to define a patients feelings of pain
• it is very subjective because pain is an emotional feeling
• the questionnaire demonstrates the difficulty associated with studying pain

Question 9

what is a key factor an experimenter must consider when carrying out animal experiments ?

Answer 9

the animals sensitivity will be affected by stress and therefore you have to ensure the animal likes you to prevent stress affecting the results

Question 10

what procedures are used to induce animal models of neuropathic pain ?

Answer 10

spinal nerve ligation- this is when one of the spinal nerve (L4,5,OR 6) is cut
partial sciactic ligation- cutting or constricting the sciatic nerve
chronic constriction injury- tying nerves up to restrict blood flow
spinal nerve injury- 2 of the 3 branches of the sciatic nerve are cut- generally the common peroneal and tibial nerves are cut and the sural nerve is left in contact

Question 11

how is a model trigeminal pain carried out ?

Answer 11

the animal is give a nice sugared drink from a tube to enable the animal to get use to drinking from it
then you inject something into the brisal area which may change their sensitivity and then you count the feediing event
e.g because if it hurts it will have increases sensitivtiy and the animal will feed less

Question 12

for electrophysiology what is an acute skin nerve preparation?

Answer 12

prep is a small piece of skin with part of a nerve attached
- can record the activity in the nerve and apply different types of stimul (thermal, mechanical and chemical) to the skin and measure the responses

Question 13

what is an in vivo electrophysiology recording used in mice models ?

Answer 13

this is more sophisticated compared to the other electrophysiology experiments

• the animal is anaesethised and then you record nociception in the cortex or DRG or spinal cord
• pinch and brush stimulus of the paw

Question 14

what experiments can be used to measure changes in neuronal gene expression ?

Answer 14

RT-PCR
in situ hybridisation
microarrays/HTS
these can be used to see how genes change in chronic pain states

Question 15

what is immunocytochemistry used for in pain studies ?

Answer 15

it can be used to characterise changes in neuronal protein expression
can used indirect antibody method
stain for proteins of interest and markers of different neurones - can determine how the change in the pain states

Question 16

what are retrograde flurorescent tracers used for ?

Answer 16

they are added at the time of legation to act as a marker to look at neuropathic pain
- after neuropathic pain develops the DRG and the nerve can be extracted and then you can determine which nerves were damaged

Question 17

what genetic manipulations can be carried out in pain research ?

Answer 17

• nociceptor specific gene deletion - this will only affect a population of DRG neurones
• siRNA gene knockdown- in vivo you can inject siRNA into DRG and suppress expression of genes

Question 18

what happened when siRNA knock down of Ltype calcium channels was carried out intrathecally ?

Answer 18

it alleviates neuropathic pain

Question 19

what are the advantages of using siRNA ?

Answer 19

• quick localised knock down of virtually any gene in adult animals therefore no developmental problems
• internal negative control e.g. if you inject at l5-l6 the input form the hind paws will be affected by the front paws will not be

Question 20

what are the disadvantages of siRNA ?

Answer 20

efficiency of knock down is hard to control
affects spinal cord and DRG- you will never know whether the effect is central or peripheral
effects wear out relatively quickly

Question 21

what happens if the KV7.2 subunit is decreased?

Answer 21

decrease in expression coincides with development of hyperalgesia
there was a partial cut of the sciatic nerve and this developed thermal and chemical hyperalgessia over a month

Question 22

what did immunocytochemistry demonstrate in mice when there was a partial cut of the sciatic nerve in relation of potassium channel Kv7.2?

Answer 22

the levels of Kv7.2 had decreased after 15 days however there was an even greater decrease after 30 days

Question 23

what transcription factor can suppress kv7 channel expression ?

Answer 23

REST
overexpression of REST decreases the expression of KCNQ channels and it produces over excitable neurones due to less expression of potassium channels

Question 24

what does peripheral neuropathic injury stimulate ?

Answer 24

stimulates expresion of transcriptional factor REST

Question 25

how does REST cause its affects to potassium cahnnels ?

Answer 25

it binds to KCNQ (kv7 gene) promoter region and suppresses its transcription and this leads to over excitable state of neuropathic nerves

Question 26

what channels in sensory neurons drve resting membrane potential towards hyperpolarizing voltages ?

Answer 26

potassium channels are th only channels