Genetic Studies
Genetic approaches to anesthetic mechanisms involve the alternatives
of forward versus reverse genetics.[166]
In forward
genetics, untargeted mutations are randomly generated and are chosen for study because
they produce lines of animals that are markedly sensitive or resistant to anesthetics.
In reverse genetics, the focus is on a particular gene that is thought to be important
in the production of anesthesia, with mutations in a cloned copy of the gene resulting
in organisms that can be tested for altered anesthetic sensitivities. Most genetic
studies have involved three different types of organisms: nematodes (Caenorhabditis
elegans), fruit flies (Drosophila), and
rodents. Nematodes and fruit flies are advantageous in genetic studies because of
their well-mapped genomes, defined nervous systems, short life cycles, and relatively
low cost. However, the behavioral assays used to assess anesthetic potencies in
these simpler organisms may be difficult to relate to the anesthetic-induced immobility
and amnesia in patients during surgical anesthesia.
Caenorhabditis elegans
The potencies of inhaled anesthetics in wild-type C.
elegans parallel those found in higher animals.[167]
Although certain behavioral end points (e.g., mating, chemotaxis, coordinated movement)
are disrupted at clinical anesthetic concentrations, anesthetic-induced immobility
of
nematodes requires anesthetic concentrations of about 5 to 10 MAC in humans.[167]
Mutants of C. elegans have been developed that are
hypersensitive to clinical anesthetics, with the genes involved encoding a homolog
of the human protein stomatin (i.e., controlling sodium and potassium flux across
membranes) or encoding a subunit of the electron transport chain.[167]
Other mutations in C. elegans in genes that encode
presynaptic proteins that regulate transmitter release produce resistance to inhaled
anesthetics.[168]
The similar properties of nonimmobilizing
agents (see Fig. 4-10
) in
C. elegans and mammals support the use of C.
elegans as a model for the study of anesthetic action.[169]
Drosophila
Potencies of inhaled anesthetics in wild-type Drosophila
(measured by response to mechanical or heat stimuli or disruption of coordinated
movement) approximate those required for surgical anesthesia. Mutants have been
obtained that are resistant and sensitive to various anesthetics,[170]
[171]
with anesthetic sensitivity depending on the
agent examined and the end point measured.[171]
Mutations that affect ion channels change the sensitivity of Drosophila
to volatile anesthetics in a manner suggesting an agent-specific action through different
neuronal pathways. For example, genetic inactivation of one class of potassium channel
decreases the potency of halothane in a brain circuit of Drosophila.
[172]
Rodents
A naturally occurring variability in anesthetic potency exists
among strains of mice and rats. The antinociceptive effect of nitrous oxide and
the development of tolerance vary markedly among different strains of rats.[165]
Anesthetic potencies (i.e., MACs) of a given anesthetic (i.e., desflurane, isoflurane,
or halothane) measured in 15 mouse strains differed by 39% to 55%.[173]
Such findings imply that multiple genes underlie the observed variability in anesthetic
potency[173]
but do not identify the genetic or
biochemical differences between strains that are responsible for the differences
in anesthetic requirement.
With selective breeding, use is made of the fact that the anesthetic
requirement varies slightly among animals of a given species and that members resistant
or vulnerable to anesthesia may be found in a normal population. Mice have been
selected from a normal population with consistently high and consistently low nitrous
oxide righting-reflex ED50
.[174]
Repeating
the process of selection, breeding, and testing for the nitrous oxide requirement
through 15 generations produced two lines of mice with requirements separated by
as much as 1 atm ( Fig. 4-15
).
[8]
[174]
Mice
resistant
to nitrous oxide also have a higher requirement for other inhaled anesthetics, but
the separation in righting-reflex ED50
values between the two lines is
inversely related to the lipid solubility of the anesthetic.[8]
The differences in nitrous oxide requirement between these lines of mice could not
be explained by an alteration in synaptic membrane fatty acid, phospholipid, or cholesterol
composition[174]
but is associated with a higher
brain norepinephrine content in resistant compared with susceptible mice.[175]
An alternative genetic approach is to knock out a gene in embryonic
stem cells thought to be important for the
Figure 4-15
The nitrous oxide righting-reflex 50% effective dose
(ED50
) values for male (closed symbols)
and female (open symbols) offspring of mice selectively
bred for resistance (HI group, circles) or susceptibility
(LO group, triangles) to nitrous oxide anesthesia.
The nitrous oxide requirements for HI and LO mice became progressively more separated
over 15 generations of selective breeding. Standard errors about most points are
less than 0.03 atm. Values for generations 1 through 10 are taken from references
[8]
and [174]
.
anesthetic state and examine anesthetic sensitivity in mice that lack functional
genes. Targeted disruption of the neuronal nitric oxide synthase gene has no influence
on isoflurane MAC or isoflurane righting-reflex ED50
in knock-out compared
with wild-type mice.[84]
Unlike wild-type mice,
acute administration of nitric oxide synthase inhibitors does not decrease anesthetic
requirement in these knock-out mice, suggesting that other non-nitric oxide mechanisms
exist in the knock-out mice to compensate for the mutation.[84]
Mice lacking the neuronal isoform of the protein kinase C gene demonstrate a small
increased resistance to isoflurane but not to halothane or desflurane.[176]
The sevoflurane requirement (i.e., MAC) is 20% lower in mice that lack the μ-opioid
receptor compared with wild-type control mice.[177]
Knock-out mice devoid of functional α2A
-adrenoreceptors exhibit
reduced isoflurane antinociception, indicating that this receptor subtype at least
partially mediates isoflurane antinociception.[61]
Mice lacking a particular
glutamate receptor subunit (i.e., GluR2) have a decreased requirement for volatile
anesthetics to produce loss of the righting reflex, whereas MAC values are unaltered
in these animals.[178]
Mice with glycine receptor
subunit mutations may be more sensitive or resistant to volatile anesthetics, depending
on the anesthesia end point and anesthetic examined.[179]
Gene knock-out of the α6
-subunit of the GABA receptor does not
alter MAC or righting-reflex ED50
for enflurane or halothane, implying
that this particular subunit of the GABA receptor is not important in the behavioral
responses to volatile anesthetics.[180]
Mice that
lack the β3
-subunit of the GABAA
receptor demonstrate
higher MAC values for enflurane and halothane but no change in righting-reflex ED50
for these anesthetics.[181]
The similar enflurane
sensitivity in spinal cords isolated from mice lacking the β3
-subunit
of the GABAA
receptor compared with wild-type controls and the decreased
role of GABAA
receptors in mediating the actions of enflurane in the mutant
mice[182]
point out a limitation in the use of knock-out
mice. Results from knock-out mice can be difficult to interpret, because mice may
adapt to the absence of a gene product by altering the amounts of other receptors
or neuroregulators that influence anesthetic requirement. Future genetic approaches
to the study of anesthetic mechanisms will involve the use of conditional gene knock-outs
selective to specific brain regions or developmental stages and a knock-in technique
to introduce a mutation into a specific receptor subunit that alters anesthetic requirement
without altering other physiologic functions.
