BJA/RCoA Non-clinical PhD Studentships

Background context
We study anaesthetic effect on carotid body oxygen sensing, hypothesising that TASK channels are involved. Patients undergoing anaesthesia are at risk of 'hypoxia' (fall in body's oxygen levels) due to partial lung collapse, blood flow derangements, drug effects, etc. This is potentially dangerous; hypoxia remains the commonest cause of anaesthetic-related death. When awake, hypoxia triggers a reflex originating in the carotid body (the chemoreflex') through which breathing is increased: this limits the fall in oxygen levels. However, anaesthetic drugs impair this reflex and reduce the patient's ability to elicit a strong reflex breathing response to hypoxia. This effect lasts well into the recovery period because low doses of anaesthetic (sufficient to blunt the reflex) are present in the body for several hours after surgery. These ideas are not relevant to this proposal but form the context to our serendipitous observation.

Novel results
In the course of these experiments, we observed that whereas halothane completely abolished hypoxic responses, isoflurane only partially did so, even at very high concentrations. In pharmacological terms halothane could be described as a 'full' agonist' (A) and isoflurane as a 'partial agonist' (B). Conventional pharmacology predicts that in a mixture of full and partial agonist, the latter antagonises the former (i.e. the effect of the mix is

Current paradigm
It is widely held that anaesthetic agents given in combination act additively. Thus if concentration A of agent A results in effect A, and if concentration B of agent B results in effect B, then a mixture of agents in concentration A+B results in effect A+B. The net result is not

Potential
This work has no immediate or even anticipated clinical relevance. However, it has potential to influence our ideas about anaesthetic mechanisms very fundamentally. If our initial results are correct, a full re-assessment of anaesthetic pharmacology will be necessary. This work is therefore about ideas and pursuit of knowledge. In future, this new understanding might drive development of targeted drugs: i.e., anaesthetics that do not have adverse effects on the respiratory system; or specific antagonists for the respiratory or anaesthetic effects. Anaesthesia might one day be reversed not by waiting for the drug to be cleared from the body, but by administering a counter-drug.

Introduction:
Moderate to severe persistent pain is a debilitating condition affecting millions of people worldwide. Opioid analgesics such as morphine are widely regarded as the best drugs for treating severe pain. They are often prescribed to patients suffering from short term pain associated with trauma and those suffering from longer term pain associated with cancer. However, opioids are not usually recommended in the treatment of persistent non-cancer pain, because their actions "wear off" due to the development of tolerance leading to a requirement for increasing opioid doses for adequate pain control. This increases the likelihood of adverse opioid events. Detrimental effects of opioids include respiratory depression, constipation and addiction. The fear of these complications often results in inadequate medication of persistent pain - this is widely acknowledged as one of the greatest unmet clinical needs. Despite intensive efforts to develop new drugs there are none available to replace opioids. We recently discovered in mice that a class of drug known as c-Src inhibitors reduces analgesic tolerance to negligible levels when given with morphine. Circumstantial evidence suggests that this is through inhibition of a pathway also involved in the development of opioid-induced constipation and respiratory depression, but not analgesia. The anti-leukaemia c-Src inhibitor, dasatinib, attenuates the development of morphine analgesic tolerance measured using a simple tail withdrawal assay of nociceptive pain in mice. The proposed research will test the hypothesis that c-Src inhibition reduces side effects associated with opioid analgesia.

Methods:
Behaviour measurements of nociceptive and affective components of pain in mice will be used to assess analgesia and tolerance by the opioids, morphine and fentanyl. Behavioural approaches will also be used to monitor constipation and respiratory depression. Mice lacking μ opioid receptors and β- arrestin2 will be used to confirm a role for these proteins. The c-Src inhibitors will be administered prior to the opioid analgesics to determine their capacity to inhibit the side effects. In vitro biochemical and electrophysiological assays will be performed to explore the effects of opioids and c-Src inhibitors on activation of c-Src and opioid receptor desensitization.

Anticipated outcomes:
It is anticipated that c-Src inhibitors will attenuate side effects of opioid analgesia. Furthermore, if the hypothesis is correct, recently developed opioid analgesic drugs that are less prone to causing side effects in mice may cause less c-Src activation and μ opioid receptor desensitization.

Conclusions:
If dasatinib alleviates the detrimental effects of analgesic opioids this will provide preclinical evidence for the initiation of trials to treat persistent pain. A positive outcome might provide sustained pain relief for millions of sufferers.

Background:
Kidney transplantation remains the first-line definitive treatment for end stage renal failure patients. However, in the UK, the scarcity of grafts and long waiting time have pushed for "marginal" graft transplantation in the last decade, wherein approx. 70% kidney grafts are from deceased donors. Unlike grafts from living donors, "marginal" grafts have diminished blood flow and oxygenation and acquire considerable tissue damages. Upon transplantation, the injured graft will not only jeopardise kidney function, it can have grave repercussions on other vital organs. It is anticipated that the extra-kidney injuries from marginal kidney graft can significantly impair the overall recovery and morbidity of patients. There is urging needs to better understand the injurious interaction between kidney and other organs, and to identify novel therapeutics that can remedy for multi-organ dysfunctions from graft deficits.

Aims:
Our first aim is to ascertain the development of extra-renal injuries (lungs and gastrointestinal tract) following marginal kidney transplantation. We will focus on specific cell death and inflammation pathways, known as necroptosis and sterile inflammation, which have been implicated in solid organ diseases. The second aim is to answer whether there is a causal relationship between necroptosis and sterile inflammation in our disease model. Our final aim is to assess whether blocking necroptosis in recipients is effective in treating lung and gastrointestinal inflammation and injury after "marginal" kidney transplantation. Such aims would reveal new drug targets in the prevention/treatment of transplant related organ dysfunction.

Methodology:
This is a pre-clinical, laboratory study consists of animal and cell culture experiments. We will use a rat model of injurious kidney transplantation to mimic marginal renal transplant in clinical situations. Rats will be divided into non-surgery control, surgery control, injury (two injury modalities) and injury + treatment (three treatment types for each of the injury modality) groups. Across all groups, different molecular markers will be measured in lungs and intestines, wherein increases strongly support development of necroptosis and sterile inflammation, and decreases in said markers would suggest resolution of injury in the treatment groups.

For cell culture study, lung and intestinal cells will be triggered to undergo necroptosis, and release of signaling molecules and concurrent inflammation will be quantified. We will then chemically inhibit or genetically ablate necroptosis, such that declines in the levels of released molecules and inflammatory components would suggest a causal relationship between necroptosis and sterile inflammation.

Expected Outcomes:
We expect marginal kidney transplantation to induce necroptosis and sterile inflammation in the lungs and small intestines. We also expect that necroptosis as a "first-hit" event can trigger "secondary" sterile inflammation, with molecules released from dying cells acting as messengers to induce inflammation of the neighbouring cells. It is further expected that blocking necroptotic cell death is effective in preventing or treating marginal transplant associated multi-organ injury.

Implications:
Our work will bring clinical awareness to multi-organ complications following marginal kidney transplantation, and further improve patient recovery during post-transplant period. Such is particularly relevant and timely in view of NHS's resolve to increase deceased organ donations and implement novel graft preservation strategies. Our study will also provide insights into molecular interactions between necroptosis and sterile inflammation to guide basic researches studying mechanisms of necroptosis.