In contrast, both anaesthetic gases (given for 60 min and without subsequent capsaicin injection) induced ERK1/2 activation in a different group of mainly lamina I neurons bilaterally. The total number of spinal dorsal horn neurons labelled on the ipsilateral side following capsaicin injection into the isoflurane-, or sevoflurane-, anaesthetised animals was significantly less than that produced by capsaicin alone. Further, capsaicin injection into isoflurane-, or sevoflurane-, anaesthetised animals reduced pERK1/2 induced by the gases alone on both sides. These key original findings are inconsistent with the suggestion that isoflurane-, or sevoflurane-, induced sensitisation of TRPV1 by capsaicin, or other agonist, is translated into induction of spinal nociceptive processing and consequential pain sensation.

In the early development of brain, there is an important period called the synaptogenesis period (also known as the brain growth-spurt period) which occurs in different mammalian species at different times relative to birth. In rodents, it begins a day or two before birth and ends 2 weeks after birth, whereas in humans it starts at the beginning of the third trimester and ends several years after birth. This is a critical period during neural development as neurons are establishing communications with their environment and other neurons to ensure that they develop and function appropriately; disruption at this stage by toxic agents including anaesthetic agents may compromise the normal neuronal development of the central nervous system. During the course of my PhD study, I found that inhalational agents isoflurane (ISO) alone or in combination with nitrous oxide (N2O) caused a wide spread neurodegeneration in the brain including the cingulated cortex, hippocampus, substantia nigra and basal forebrain in postnatal day 7 Sprague-Dawley rat pups. In those brain regions, the cells involved included glutamatergic, GABAergic and dopaminergic neurons. However, the cholinergic neurons in the basal forebrain were spared from anaesthesia-induced neuroapoptosis. My studies also showed that the neurodegeneration was not only confined to the higher centres, but was also observed in the spinal cord. These data indicated that the anaesthetics studied acted at molecular level and induced neuronal apoptosis by activating the intrinsic apoptotic pathway. It has been well documented that preconditioning with hypoxia or other interventions including pharmacological agents can protect against subsequent brain injury induced by hypoxia/ischemia. However, my data showed that hypoxic preconditioning exacerbated N2O+ISO-induced neonatal neurodegeneration, while xenon preconditioning protected against anaesthetics-induced neuroapoptosis and subsequent neurocognitve impairment. Most studies in this field including my own study described above are more relevant to the clinical situation where neonates are managed in the critical care unit, i.e. in the absence of surgery. For this reason, I implemented another experimental setting to mimic the clinical situation in the operating room where neonates receiving anaesthetic drugs are also subjected to nociceptive surgical stimuli. Therefore, in another experiment, rat pups in addition to receiving anaesthetic drugs were also subjected to standardised nociceptive stimuli. The results suggested that nociceptive stimuli enhanced neuronal apoptosis induced by anaesthetic drugs in the brain and the spinal cord and consequently impaired neurological function. Were the data reported in my thesis to be extrapolated to the clinical setting, it would have very important implications for the clinical management of paediatric population receiving general anaesthesia.

TASK channels, members of the two-pore-domain potassium channel family, contribute towards the resting membrane potential and have been implicated in the mechanism of general anaesthesia. Previous work from our group with a TASK-3 channel knockout (T3KO) mouse showed a reduction in halothane sensitivity using a loss of righting reflex (LORR) assay, and absence of the theta brain oscillation induced in wild type (WT) mice by halothane anaesthesia. Two further strains of knockout mice, the TASK-1 knockout (T1KO) and the double knockout (DKO: TASK-1 and -3 channels), were compared with WT using the LORR assay, cortical electroencephalogram recording in response to halothane and during sleep. The mechanistic basis for the diminished theta oscillation in T3KO mice was investigated by recording in CA1 pyramidal cells of the hippocampus. The LORR assay revealed a decrease in halothane sensitivity in T1KO but not DKO compared with WT. The T1KO strain had a theta oscillation induced by exposure to halothane similar to that of WT mice, whereas that observed in the DKO was intermediate between WT and T3KO. T1KO differed from other strains in that the distribution of sleep and wake periods was uniform across the diurnal cycle. The resting membrane potential did not differ between strains during control or halothane exposure. During control there was no strain difference in action potential (AP). Halothane altered AP shape in WT but not the T3KO strain. WT had a greater ability to sustain AP firing than T3KO during halothane. These data show that T1KO mice have decreased anaesthetic sensitivity and altered sleep structure compared with WT, indicating a role for this channel in anaesthetic sensitivity and sleep. The similar resting membrane potential and lack of response to halothane in the T3KO makes pyramidal cells an unlikely source of the theta ablation observed.

Stroke in general but especially in the postoperative period is a serious clinical problem that warrants new therapeutic approaches. Here neuroprotective strategies and especially preconditioning have recently emerged as promising. Preconditioning was originally demonstrated in the heart but was subsequently also found in other organs. Classically it describes a phenomenon where short periods of ischaemia render tissues less vulnerable to major infarcts. In addition to ischaemia neuronal preconditioning can be achieved pharmacologically as well as through inhalational anaesthetics or drugs that open ATP-sensitive K+(KATP)channels. However, the mechanisms through which anaesthetics produce protection remain elusive and the use of K+ channel openers is hampered by their inability to cross the blood-brain-barrier. This study was conducted to investigate the effects of inhalational anaesthetics on KATP channels and to explore whether their neuronal preconditioning properties were dependent on KATP channel opening. First, in whole-cell and excised patch clamp experiments the effects of inhalational anaesthetics on recombinant wild type neuronal (Kir6.2/SUR1) KATP and related as well as modified channels were evaluated. Secondly, the KATP channel dependence of anaesthetic preconditioning was tested in neuronal-glial co-cultures. Recombinant KATP channels were activated by xenon, but inhibited by halogenated volatiles. Moreover, it was shown that xenon acted directly on the Kir6.2 pore-forming subunit, reduced the ability of ATP to inhibit the channel and had no effect on the ATP-regulated Kir1.1 channel. Functionally both sevoflurane and xenon preconditioned neurons at clinically used concentrations but only the effect of xenon was dependent on KATP channel activation. Thus this study established xenon as a novel KATP channel opener. It interacts with the pore-forming Kir6.2 rather than the regulatory sulphonyl urea receptor subunit and disinhibits the channel from the blocking actions of ATP. As a consequence xenon but not sevoflurane is able to precondition neurons in a KATP channel-dependent manner.

The last 20 years have seen a substantial advancement in the understanding of the molecular targets of general anaesthetics however the neural mechanisms involved in causing loss of consciousness remain poorly understood. Thalamocortical oscillations are present in natural sleep and are induced by many general anaesthetics suggesting that modulation of this reciprocal system may be involved in the regulation of consciousness. Dynamic changes of thalamocortical oscillations in natural sleep and anaesthesia were investigated in rats chronically implanted with skull screw and depth electrodes in the cortex and thalamus. The hypothesis that discrete areas within the thalamus are responsible for regulation of arousal was tested. The anaesthetics propofol and dexmedetomidine but not midazolam produced switches in delta frequency at loss of righting reflex (LORR). This switch in frequency mirrored that seen within non-rapid eye movement sleep (NREM), whereas the onset of NREM was characterized by a switch from theta to delta in the EEG. Depth recordings during NREM indicated that the switch into a NREM state occurred in the central medial thalamus (CMT) significantly before the cingulate, barrel cortex and ventrobasal nucleus (VB), and that the CMT switch corresponded to the switch seen in the global EEG. Dexmedetomidine hypnosis showed a delta frequency shift that occurred simultaneously within the thalamus and cortex, and furthermore that the thalamus exhibited phase advancement over the cortex at the point of LORR. In conclusion, globalised changes within the thalamocortical system occur for propofol and dexmedetomidine LORR in the rat. This change represents a transition within drug free NREM and may implicate a common pathway responsible for a decrease in arousal. Furthermore, the phase advancement of the intralaminar thalamus over the cortex at LORR suggests a crucial role for this part of the thalamocortical system for regulating consciousness.

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