Theories of Anaesthesia

1. Theories of Anaesthesia Dr. Pratheeba Durairaj,MMC 14.11.08

2. Introduction • The mechanisms of anesthesia are surprisingly little understood • Anesthetics are unique drugs in pharmacology. They affect all macromolecules. • The diversity of the structures of these molecules indicates that there are no common receptors. • The action of anesthetics is nonspecific and physical

3. Why should we search for mechanisms of anaesthesia ? • The safety of anaesthetics has improved over the years even though we still are to find exactly how they act. • Search of molecular mechanisms may lead to development of safer drugs with less adverse effects

5. Cell fat dissolution theory • Von Bibra and Harless, in 1847, were the first to suggest that general anaesthetics may act by dissolving in the fatty fraction of brain cells. • They proposed that anaesthetics dissolve and remove fatty constituents from brain cells, changing their activity and inducing anaesthesia.

6. Colloid theory • 1875 – CLAUDE BERNARD • Studied anaesthetic induced inhibition of protoplasmic streaming in slime moulds • Proposed that a reversible coagulation of cell colloids accompanied anaesthesia

7. Meyer and Overton hypothesis • 1899 /1901 - Lipid solubility theory - H. H. Meyer / Overton. • The most striking correlation observed 100 yrs ago between the physical properties of general anaesthetic molecules and their potency. • States that narcosis occurs when critical drug concentration is achieved in crucial lipid of CNS • Suggests that when an anaesthetic dissolves in a liphophilic portion of membrane, blockade of essential pore ? Sodium channel occurs preventing depolarisation

8. Contd… Meyer • compared the potency of many agents, defined as the reciprocal of the molar concentration required to induce anaesthesia in tadpoles, with their olive oil/water partition coefficient. • Nearly linear relationship between potency and the partition coefficient for many types of anaesthetic molecules. • The anaesthetic concentration required to induce anaesthesia in 50% of a population of animals (the EC50) was independent of the means by which the anaesthetic was delivered, i.e., the gas or aqueous phase. • These results on lipid-free proteins show that the correlation between lipid solubility and potency of general anaesthetics is a necessary but not sufficient

10. LIMITATIONS • Only applies to gases and volatile liquids –oil gas partition coefficient can’t be determined for liquid anaesthetics • Olive oil is a poorly characterized mixture of oils

11. Exceptions • From the Meyer-Overton correlation, alcohols should become increasingly potent as the carbon chain length increases because the alcohols grow more hydrophobic. • Instead of becoming increasingly potent without limit however, at certain chain lengths the addition of just one methylene group causes the molecule to lose its ability to anaesthetise. • Decrease in anaesthetic potency in higher members of homologous series is CUTOFF EFFECT.[eg] . n-pentane causes anaesthesia but not n- decane • STEREOISOMERS • Enflurane and Isoflurane, are isomers with identical chemical properties [oil gas partition coeffecient], but can differ greatly in anaesthetic potencies. –suggest that potency depends on factors other than lipid solubility • CONVULSANT GASES • Some lipid soluble compounds are convulsants and not anaesthetics

12. Altered Cell Membrane Permeability • 1907/1909 –HOBER AND LILLIE • Suggested absorbed anaesthetics decrease cell permeability • Cell was rendered less capable to undergo depolarization and thus inhibited • Turned out to be precedent of a modern theory of protein receptor

13. Membrane volume expansion • 1954 –MULLINS • Proposed that potency correlated better with volume rather than number of anaesthetic molecules dissolved in oil phase of membrane • Speculated that they fill voids in membrane rather than adding to membrane’s volume

14. Alteration of lipid layer fluidity • 1968 –Metcalf et al • Demonstrated that Benzyl Alcohol increased mobility of membrane components of erythrocytes • Suggested that anaesthetics act by increasing membrane lipid fluidity which in turn perturbs membrane protein function

15. Pressure Reversal of Anaesthesia The observed pressure reversal of anaesthesia and narcosis is one of the most intriguing features of the anaesthetic state. –DOCUMENTED OVER 50 YRS Two theoretical explanation for this effect. • Anaesthetics and pressure act on different molecular targets • The pressure reversal is a mere consequence of a general stimulation brought on by pressure overcoming the general depression of physiological and mental activity caused by the anaesthetics • Anaesthetic potencies of various substances are similar among a wide range of organisms, the values of the pressures required to reverse anaesthesia vary considerably between organisms, and in some cases the pressure reversal is not observed at all.

16. An alternative point of view assumes that pressure and anaesthesia act antagonistically at the same molecular sites, The pressure reversal effect is intimately related to a general mechanism of anaesthetic action. • Most obvious effects of the increase of pressure at constant temperature is a volume reduction some authors have argued that anaesthetics act by increasing the local volume contribution of some crucial target in the nervous system. That is the basis of the critical volume hypothesis .

17. In view of the lipid theories, the pressure reversal effect is a consequence of the observation that higher pressures reverse many anaesthetic-induced perturbations of lipid bilayers. • Protein theories, that assume direct binding of anaesthetics to protein sites within ion channels or at receptor sites, account for pressure reversal through dislocation/dissociation of the anaesthetic molecules from their usual targets, or modification of the target action

18. Critical volume hypothesis • 1973 –MILLER • Based on theories of lipid solubility & pressure reversal of anaesthesia ,he postulated that anaesthesia occurs when they expand membrane volume beyond a critical amount by 1.1 % • Changes in membrane volume compresses ion channel and alters function • Increases in membrane thickness alters neuronal excitability by changing potential gradient across the membrane

19. Phase Transition Theory • 1977 – TRUDELL • Speculated that during membrane excitation ionic channel protein under go conformational changes – increases lateral dimensions of protein and opens ionic channel • Normally, membrane lipid near ionic channel exists in fluid state Compact Gel state during depolarization • Small increases in membrane fluidity – large decrease in lateral compressibility of bilayer – prevent conformational changes in ionic channel –inhibits membrane excitation • Anaesthetics enhance membrane disorder by increasing fluid to gel ratio –interfering with ability to open/close channels

20. Lipid theories • The Meyer–Overton observations - earliest and still the best correlation • Those findings led to a general theory that anaesthetics dissolve in the lipid fraction of the cell membrane, thus altering the physiological properties. • Modern variants of the lipid theory developed related membrane properties that could be relevant to anaesthesia, including volume expansion and lateral surface pressure, membrane fluidity and thickness, and surface tension effects.

21. Such an altered state of the membrane lipids might then change the activity and function of integral membrane proteins, including ion-channels, thereby inducing anaesthesia. • Modern lipid theories often postulate such an indirect mechanism for the occurrence of anaesthesia/narcosis. • The main drawback of these models lies in the observation that theanaesthetic concentrations needed to produce relevant changes in membrane lipid properties would be highly toxic to the organism.

22. Macromolecule–water interface theories • Many of the gas phase species that exhibit anaesthetic properties also form crystalline hydrates. • PAULING proposed that hydrated anaesthetic gas molecule [clathrate ] can stabilize a membrane / occlude essential pores- interferes with depolarization • MILLER postulated that interaction between water and anaesthetic molecule results in an iceberg which stiffens up membrane & prevents neuronal transmission • Anaesthetic gas molecules might then occupy structure-determining cavity sites within dynamic ice-like liquid water clusters.

23. Limitations • Neither Clathrate nor iceberg can be formed in ambient pressure and body temperature • Typical gas clathrate hydrates are not stable under physiological pressure–temperature conditions. • Additional factors were considered that could increase the clathrate stability in a physiological environment

24. Protein Theories • Go back to the late 19th century • The remarkable finding in1993 –Franks and Lieb that the soluble protein of firefly luciferase - a good model for anaesthetic action represented a key advance in the field. • Detailed analysis of anaesthetic–luciferase interactions led to the suggestion that anaesthetic molecules compete with substrate luciferin molecules for binding to the protein hydrophobic pocket.

25. Contd… • It is now thought that ion channels and neurotransmitter receptor sites formed from protein complexes embedded within the neuronal cell membranes constitute the primary sites of anaesthetic action. • Discovery of the stereospecificity of certain anaesthetics and their optical isomers, which are equally soluble in lipids, supports a protein-based theory of anaesthetic action. • Opiates can be antagonized by naloxone, benzodiazepines by flumazenil and non-depolarizing muscle relaxants by neostigmine or edrophonium. • Therefore, in theory it should be possible to antagonize i.v. anaesthesia induced by a combination of opiate, benzodiazepine and muscle relaxant by giving acombination of antagonists for each substance.

26. Contd… • No effective antagonist for inhalation anaesthesia has been reported so far. This does not mean that anaesthesia cannot be brought about by receptor action. • Inhalation anaesthetics may simply act at several receptors that differ in their pharmacodynamic properties and require several different antagonists that have still not been found

27. Neurophysiological Theories • Suggest that synapses is a likely site of action • Increase in synapses increases anaesthetic sensitivity • Mechanism of action –change in calcium permeability at susceptible synapses – decreased release of neuro transmitter • Inhalational anaesthetics affect membrane structure -decreasing ability of sodium channels to open • LIMITATIONS • Does not explain how they act • Changes in EEG & SSEP in man differ widely for different anaesthetics suggesting multiple sites of action • RAS Is not the sole site • Only an anatomical description rather than molecular expression

28. Biochemical theories • Volatile agents inhibit mitochondrial respiration in a reversible and concentration dependent fashion • Do produce many biochemical effects -Alteration in flux of calcium across mitochondrial and neural membranes -Increase the concentration of GABA in synaptic areas by inhibiting degradation-anaesthetic modulation

29. Molecular and cellular theories • Defined as those theories that are based on anaesthetic mechanisms at the molecular or cellular level that has been proposed to be responsible for generating the state of general anaesthesia. • Enhancement of activity at GABAA receptors is an important component, perhaps the only component, of the mechanisms that are relevant in anaesthesia

30. Genetic studies • Genetic manipulation of animals –increasingly used for investigating links between potential targets of anaesthesia and their behavioral effects • Screening for mutations in fruit flies & nematodes – identified strain with altered sensitivity to anaesthetics but applicability to human studies is questionable • Introduction of specific mutants into native genes [knock in] enables assessment of physiological & pharmacological role of specific proteins • This method provided evidence for involvement of β2 & β3 sub units of GABA A receptor background leak potassium channels in producing immobility ,sedation &hypnosis • Highlighted that GABA A – β subunits are partially responsible for volatile anaesthetic action but crucial for IV agents like Propofol & Etomidate • Genetic studies on Drosophilaeven point to voltage-gated sodium channels as factors that may affect anaesthetic sensitivity

31. Integrated theories • Integrated theories of general anaesthesia are theories that do not treat the several components of general anaesthesia independently but provide an integrating basis for their explanation.

32. Targets of Anaesthetic Action • Molecular targets • Depending on the physicochemical nature of the anaesthetic ,they may prefer Within the bilayer, the interface between the lipid and the aqueous phase Between the lipid and the membrane protein Hydrophobic core of the lipid bilayer itself Protein-binding sites exposed to the aqueous phase Inside membrane proteins or in the lumen of ion channels Within aqueous domains or water channels.

34. Contd… • May bind within the core of the membrane protein itself, between hydrophobic -helices and hydrophobic or lipophilic pockets • May interfere in the interactions between subunits of a protein or between different proteins • Interest is focused on anaesthetic interaction sites that are amphipathic or lipophilic, i.e. sites that have some polar components besides a hydrophobic component.

35. Contd… • All members of the main families of ion channels and their many subtypes are affected by anaesthetics Sodium channels, Potassium channels, Calcium channels (both voltage and ligand-sensitive), • Glutamate receptors [N-methyl-D-aspartate (NMDA), -amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) and kainate] • Novel P2X receptors, n AChR • 5-HT3 receptor channels, GABA A receptor channels and glycine receptor channels.

36. Voltage gated ion channels • Na+ & K+ channels - Required for action potentials • Insensitive to volatile agents [required 8 times of halo that of producing anaesthesia- HAYDEN ] • Voltage Dependent calcium channel –couple electrical activity to cellular function – relatively insensitive -inhibited by volatile agents 2-5 times required for anasthesia

37. Ligand Gated Ion Channels • Fast excitatory & inhibitory neurotransmission • Glutamate activated ion channels – • 3 types- AMPA, KAINATE - insensitive to halothane /sensitive to barbiturates • NMDA receptors - Ketamine is a potent /selective inhibitor • N2O, XENON inhibit excitatory NMDA Glutamate transmission • Glutamate receptors – Glu R3 –inhibition ,GluR6 –enhanced

38. GABA Activated Ion Channels • Barbiturates, anaesthetic steroids, benzodiazepines, propofol , etomidate ,volatile agents modulate GABA A receptor • Volatile agents alter /potentiate Ligand binding in GABA A receptor • Volume of anaesthetic binding sites on α sub unit of GABA A receptor is between 250 & 370 cubic • Xenon, Nitrous oxide and Cyclopropane, have little effect on GABA -A receptors. • Genetic studies and evidence from brain imaging do not support an exclusive role for GABA - A receptors in anaesthesia, although they support the hypothesis that the invivo effects of anaesthetics are mediated at least in part through GABAergic mechanisms

39. GABA A receptor

40. Contd… OTHERS • Volatile agents stabilize the Ach receptors in a - conformational stage – inactive state • These receptors play a role in behavioral & physiological effects • Have a agent specific effect on 5HT 3 receptors • Glycine receptor- chloride sensitive ion channel –potentiated by ? Propofol, pentobarbitol

41. Subcellular targets • Anaesthetics act on axons and dendrites and presynaptic and postsynaptic membranes as well as on the somatic membranes of neurones and glia. • They act on many intracellular structures, such as the neurotransmitter release system, the calcium homeostasis and buffering system, second-messenger cascades and mitochondria.

42. Cellular targets • Glial cells,skeletal and cardiac myocytes, endocrine cells and cells of the immune system are also targets. • Local microcircuitsAnaesthetic acts on microcircuits within slices from dorsal root ganglion,spinal cord, thalamus, hippocampus,cortex and cerebellum as well as in neuronal networks grown in culture.

43. Systems • Inhalation anaesthetics, to a greater extent than i.v. anaesthetics, affect all areas of the CNS. • Imaging studies indicate that a number of discrete brain structures are related to the effects of anaesthetics, spinal cord, brainstem, cerebellum, midbrain and thalamus, midbrain reticular formation, basal ganglia, superior frontal gyrus, anterior cingulate gyrus, posterior cingulate, basal forebrain, insular cortex, prefrontal cortex, parietal and temporal association areas, occipitoparietal association cortices and occipital cortex..

44. Contd… • Functional imaging techniques are beginning to help identify key brain structures that appear to play important roles in the different clinical endpoints produced by anaesthetics. • The peripheral nervous system also provides targets for anaesthetic actions, as does the endocrine system and the immune system.

45. Unitary hypothesis • Overton was a proponent of the unitary hypothesis when he stated that, it is highly probable that the mechanism of ether or chloroform narcosis, for example, remains substantially the same in the ganglia cells, the ciliary cells, and in the plant cells as well’.

46. Contd… • Halsey discussed the unitary hypothesis for inhalation anaesthetics (equivalent to Overton’s non-specific narcotics) three-quarters of a century later • Stated that a ‘unitary hypothesis’ of anaesthesia did not require a single gross site of action, as there was evidence for several gross sites. • For him, the unitary hypothesis required identity of action at the molecular level. • Thus, the first possible model was never seriously advocated.

47. Contd… • Another example of a unitary hypothesis is the GABA hypothesis, -- states that enhancement of activity at GABA A receptors is an important component, perhaps the only component, of relevant mechanisms in anaesthesia. • Results obtained from genetic studies are now being used as evidence that the unitary hypothesis can be dismissed in the nematode.

48. Multisite hypotheses • Allow that many molecular mechanisms may cause one or many neuronal lesions, do not hold that all anaesthetics should show the same correlation between the anaesthetic endpoint and the mechanism-related endpoint. • Thus, if certain inhalation anaesthetics do not have much effect on GABA A receptors, this does not imply that GABA A receptors are not important molecular players in the clinical components of anaesthesia. • Studies of proteins, second-messenger signalling, the spinal cord, brain slices, genetics and functional imaging all come up with data consistent with multisite theories of anaesthetic action

49. High pressure effects in anaesthesia and narcosis • X-ray crystallography has been used to investigate the incorporation of species like Xe in hydrophobic pockets within model ion channels that may account for pressure effects on neuronal transmission. • Magnetic resonance imaging techniques are providing tomographic three-dimensional images that detail brain structure and function, and that can be correlated with behavioural studies and psychological test results. • Voltage-sensitive dye (VSD) imaging studies on brain slices provide time-resolved images of the dynamic formation and interconnection of inter-neuronal complexes.

50. xenon anaesthesia • Use of Xe results in remarkable cardiovascular stability, rapid onset and offset of its action resulting from its extremely low blood–gas partition coefficient, neuroprotection and profound analgesia • Kr shows anaesthetic effects at higher pressures than Xe • Biochemical studies combined with crystallography and molecular dynamics simulations indicate that Xe, Kr and N2 could occupy hydrophobic sites or pockets within ion channels such as those associated with the excitatory glutamate (NMDA) neurotransmitter receptor complex