EXPERIMENTAL NOTES
Section B (Experimental) - answer 1 question out of a choice of 2
SKIN ASSAY
Inflammation estimated by measuring oedema formation in the skin
Effect of vasoactive agents in human skin
- Vasodilators
o Prostaglandins, nitric oxide
o Neuropeptides (CGRP)
- Mediators of increased microvascular permeability
o Histamine, Bradykinin
o LTC4, LTD4, PAF
o Substance P, VEGF
The Skin Assay: measurement of dorsal inflammation
- Provides an understanding of mechanisms involved in microvascular permeability
changes in response to inflammatory mediators (First called the Miles Assay).
o Relatively fast in vivo technique – blood vessels and cells intact
o In vivo experiment as the skin is composed of nerves, cells and blood
components, all of which can contribute to inflammation.
o In vivo skills required
- Testing ability and potency of hyperpermeability inducing/blocking compounds
- Any proposed or established mediators or their blockers can be studied
- Can extend to measure cell types (e.g. neutrophils, monocytes etc.)
- Mediators under study here are substance P and CGRP.
o CGRP more vasodilator effects – one of most potent vasodilators known
o Substance P more mediator of vascular permeability – also potent
BUT:
- Does not take into account blood pressure or flow
- Vasculature in skin, translatable to other tissues like brain, lung?
- Reversible gaseous anaesthetics used – avoid vasoconstriction which could
interfere with the assessment of microvascular permeability changes.
- Skilled experimenter
Health and Safety Aspects:
, - Allergies
o Could you be allergic?
o Wear facemask
o Be careful with fur and dander
- Pinprick injuries
o Go to cold water tap/hand wash basin, remove glove
o Press around area (to allow removal of fluid) and wash well
o DISPOSE OF NEEDLES IN SHARPS BIN
Drugs for i.d. injections
- CGRP and substance P are stored in the freezer in small aliquots (Eppendorf
tubes); unfreeze enough for each practical.
Anaesthetics
Intraperitoneal (i.p.) injection of urethane (7 g/g)
- Urethane anesthesia is characterized by a relatively long duration of action,
lasting several hours.
- It provides stable anesthesia with minimal respiratory depression and
cardiovascular effects.
- Urethane is often preferred for studies requiring prolonged anesthesia or when
repeated dosing is necessary.
Can also use isofluorane (2% in O2) anaesthetic
- Isoflurane is administered via inhalation.
- It is typically delivered as a gas mixture containing 2% isoflurane in oxygen (O2).
- Isoflurane anesthesia is characterized by rapid induction and recovery, making it
suitable for short-term procedures.
- It provides precise control over the depth of anesthesia and is easily adjustable.
, - Isoflurane may cause respiratory depression and cardiovascular effects at higher
concentrations or with prolonged exposure.
Chemical Maximum Hazard Disposal
Concentration
Peptides 10-4 Molar in Potential Pour down laboratory
(SP/CGRP) Tyrodes inflammation sink followed by lots of
water
Evans Blue 10-3 Molar in Toxic. Pour down laboratory
saline sink followed by lots of
water
Urethane 10-3 Molar in Toxic. Class Pour down laboratory
saline students sink followed by lots of
should not water
be exposed
to powder
CAN DILUTE DRUG STOCK IN TYRODES SOLUTION
We give injection volumes of 50µl intradermally into skin with a 27g needle (grey)
- Place drugs for injections into syringes
- Fill as needed, no bubbles
- Keep on ice as much as possible for stability
- Keep guard on needle before/after use (minimise risk of injury)
We shave dorsal skin of mouse
Mark out dorsal skin sites (6, 1-6)
- Balanced site design, random allocation of agents
Section B (Experimental) - answer 1 question out of a choice of 2
SKIN ASSAY
Inflammation estimated by measuring oedema formation in the skin
Effect of vasoactive agents in human skin
- Vasodilators
o Prostaglandins, nitric oxide
o Neuropeptides (CGRP)
- Mediators of increased microvascular permeability
o Histamine, Bradykinin
o LTC4, LTD4, PAF
o Substance P, VEGF
The Skin Assay: measurement of dorsal inflammation
- Provides an understanding of mechanisms involved in microvascular permeability
changes in response to inflammatory mediators (First called the Miles Assay).
o Relatively fast in vivo technique – blood vessels and cells intact
o In vivo experiment as the skin is composed of nerves, cells and blood
components, all of which can contribute to inflammation.
o In vivo skills required
- Testing ability and potency of hyperpermeability inducing/blocking compounds
- Any proposed or established mediators or their blockers can be studied
- Can extend to measure cell types (e.g. neutrophils, monocytes etc.)
- Mediators under study here are substance P and CGRP.
o CGRP more vasodilator effects – one of most potent vasodilators known
o Substance P more mediator of vascular permeability – also potent
BUT:
- Does not take into account blood pressure or flow
- Vasculature in skin, translatable to other tissues like brain, lung?
- Reversible gaseous anaesthetics used – avoid vasoconstriction which could
interfere with the assessment of microvascular permeability changes.
- Skilled experimenter
Health and Safety Aspects:
, - Allergies
o Could you be allergic?
o Wear facemask
o Be careful with fur and dander
- Pinprick injuries
o Go to cold water tap/hand wash basin, remove glove
o Press around area (to allow removal of fluid) and wash well
o DISPOSE OF NEEDLES IN SHARPS BIN
Drugs for i.d. injections
- CGRP and substance P are stored in the freezer in small aliquots (Eppendorf
tubes); unfreeze enough for each practical.
Anaesthetics
Intraperitoneal (i.p.) injection of urethane (7 g/g)
- Urethane anesthesia is characterized by a relatively long duration of action,
lasting several hours.
- It provides stable anesthesia with minimal respiratory depression and
cardiovascular effects.
- Urethane is often preferred for studies requiring prolonged anesthesia or when
repeated dosing is necessary.
Can also use isofluorane (2% in O2) anaesthetic
- Isoflurane is administered via inhalation.
- It is typically delivered as a gas mixture containing 2% isoflurane in oxygen (O2).
- Isoflurane anesthesia is characterized by rapid induction and recovery, making it
suitable for short-term procedures.
- It provides precise control over the depth of anesthesia and is easily adjustable.
, - Isoflurane may cause respiratory depression and cardiovascular effects at higher
concentrations or with prolonged exposure.
Chemical Maximum Hazard Disposal
Concentration
Peptides 10-4 Molar in Potential Pour down laboratory
(SP/CGRP) Tyrodes inflammation sink followed by lots of
water
Evans Blue 10-3 Molar in Toxic. Pour down laboratory
saline sink followed by lots of
water
Urethane 10-3 Molar in Toxic. Class Pour down laboratory
saline students sink followed by lots of
should not water
be exposed
to powder
CAN DILUTE DRUG STOCK IN TYRODES SOLUTION
We give injection volumes of 50µl intradermally into skin with a 27g needle (grey)
- Place drugs for injections into syringes
- Fill as needed, no bubbles
- Keep on ice as much as possible for stability
- Keep guard on needle before/after use (minimise risk of injury)
We shave dorsal skin of mouse
Mark out dorsal skin sites (6, 1-6)
- Balanced site design, random allocation of agents
