sodium channels and pain 3

Question 1

where is Nav1.7 expressed?

Answer 1

• in the PNS- sensory and sympathetic ganglia (very low expression in CNS)
• stronger expression in pain sensing then non-pain sensing neurons (may have function in pain modelling?)

Question 2

How can you get tissue specific deletions of Nav1.7?

Answer 2

• use cre/lox system
• cross 2 transgenic lines
• In one line target gene is floxed with lox sites at 2 exons of gene
• In second transgenic line cre recombinase is expressed downstream of a tissue specific promoter - so cre is only expressed in a certain tissue
• cre recombinase cuts lox sites on floxed gene in tissue only where cre is expressed (e.g.DRG)- specific tissue gene knockout
• can be tested that cre is expressed in DRG only - tested by staining
• larger cells not stained - smaller cells stained - what we want as smaller cells are nociceptive

Question 3

what cre and floxed specific mice were crossed?

Answer 3

Nav1.8 cre mouse
Nav1.7 floxed mice

(Nav1.7 deletion in Nav1.8 expressing cells)

Question 4

what were the pain phenotypes when Nav1.7 was knocked out in Nav1.8 positive neurons?

Answer 4

• deficit in sensing mechanical pressure
• acute pressure - resistant
• inflammatory pain - ABOLISHED (absence of inflammatory pain)
• BUT neuropathic pain is not reduced

Question 5

what does diphtheria toxin do?

Answer 5

-causes death of small (nociceptive) DRG

Question 6

How were Nav1.8 positive neurons ablated?

Answer 6

• Nav1.8 - cre mouse used to activate the expression of diphtheria toxin in small DRG only
• so only small DRG that are Nav1.8 positive die

-cells that don’t express Nav1.8 left

Question 7

what was the pain phenotype when Nav1.8 positive neurons were ablated?

Answer 7

• resistance to cold pain and acute pressure
• resistant to inflammatory pain
• BUT still no change in neuropathic pain

Question 8

when is neuropathic pain attenuated?

Answer 8

when Nav1.7 is deleted in all DRG and sympathetic neurons (all peripheral neurons)
(by using wnt1-cre mouse -deletes nav1.7 in all peripheral nerves)

(can’t delete nav1.7 from all cells as mouse dies)

Question 9

what does advillin cre mouse do?

Answer 9

-deletes floxed nav1.7 in all DRG neurons

when this was done there was no deficit in neuropathic pain

Question 10

what is caused by GOF mutations in Nav1.7 in humans?

Answer 10

-PE (primary erythermalgia)
dominant
pain triggered by standing, eating, heat
intermittent burning pain

• Familial rectal pain
  dominant
  pain when defecating

Question 11

what does loss of Nav1.7 do in the mice/humans?

Answer 11

• increase in endogenous opioid expression
• opioid antagonism by NALOXONE reverses loss of pain in KO mice

-LOF mutation causes no feeling of pain in humans (CIP?)