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Haitian (Billy) HeÌý

Supervisor: Dr. Donald van Meyel

Anyone who has gone through the recovery process from severe physical injury willattest to two facts: despite itsbriefness or lightness, contact with the affected area can be painful and further injury to the area will be excruciatingly painful. There is an analogue to these symptoms for burn victims as well. We sensitize ourselves to lower temperatures and respond more severely to painful heat stimuli. In scientific terms, allodynia is the sensitization to previously non-painful stimuli, and hyperalgesia is the hyper-sensitization to already painful stimuli. The goal of my project is to elucidate the underlying signalling mechanisms that induce allodynia/hyperalgesia. Such knowledge is immensely beneficial for developing treatments for a variety of illnesses and injuries.

One of the biggest issues in patient care is pain reduction. Allodynia and hyperalgesia usually accompany injury as critical defense mechanisms for guarding healing tissues. Under normal conditions, the appearance of both of these phenomena is actually quite useful for expediting the rate of recovery. However, in certain cases, they persist past full recovery, resulting in chronic pain. This persistent hypersensitivity is quite prevalent and leads to a wide variety of disabilities, suffering and costs to society. Current treatments, like opiate administration, often result in tolerance, dependence, nausea, etc. These limitations motivate efforts, similar to my own, to produce more efficient and safe alternative treatments.

My research involves measuring the effects of injury on Drosophila (fruit fly) larvae that we genetically modify. Larvae have a surprisingly robust sense of pain that is easily observable(1). Simply, we give larvae sunburn (controlled UV irradiation) to effect superficial tissue damage. In normal larvae, this generates a typical allodynic/hyperalgesic behavioural response – the larvae actually roll towards the painful stimulus – which we demonstrate using our custom-made heat probe. However, from our own work and others (2-4), we know that alterations to the Hedgehog signalling pathway (a prevalent signalling pathway among all vertebrates, named after the primary secreted protein Hedgehog) can modulate and even abolish this behaviour. By controlling which proteins are affected, I hope to refine or discover new aspects of the signalling system for the benefit of new treatments.

Unfortunately, there is rather large gap in our understanding of the players involved in the Hedgehog pathway in the context of pain transduction. In general, we know that without Hh binding to its receptor during and after the injury, there would be no induction of allodynia/hyperalgesia (In more physical terms, it’s analogous to being able to bump your recently healed broken arm and feeling absolutely normal). Unfortunately, to target and disrupt the Hh gene would be disastrous as it is required in a huge number of roles outside of pain sensitization, resulting in complex and unwanted side-effects. The better alternative is to be able to disrupt signalling specifically in the neurons that transmit the pain.

Through our lab’s previous work, we have shown that two proteins are necessary for active Hh signalling – Interference Hedgehog (Ihog) and Brother of Ihog (Boi) (2). So far, they seem to be doing the exact same role in every tissue system previously investigated and can compensate for one another, yet this theory is untested in the pain sensing neurons of larvae. And so, my project was born. Larvae, either having only functional Ihog or Boi or both (i.e. wild-type, as a control), would be irradiated, causing mild tissue damage in the body wall, putatively sensitizing the pain neurons that go to innervate it. Following irradiation, I would test for the presence and severity of the allodynia/hyperalgesia. Our initial hypothesis was that single mutant larvae would show reduced allodynic/hyperalgesic responses as a result of the loss of one of Hh’s binding partners, Ihog or Boi.

There were two major surprises that came along while recording the responses of the larvae. Firstly, even without UV irradiation, the single mutant larvae actually exhibited a heightening of sensitivity to thermal stimuli (Figure 1B). This runs counter to our expectations of the effects that absence of these proteins would produce. In fact, we see a response analogous to over-activation of the pathway! Moreover, as seen inÌýFigure 1B, though Boi mutants seem to generate some mild hypersensitization, Ihog mutants show dramatically increased baseline sensitivity and hypersensitization at both allodynic and hyperalgesic response ranges. This gives Ihog an unprecedented new distinct role separating it from Boi.

In total, these preliminary results hint at potential new signalling pathways apart from the canonical Hh pathway. Foremost, there is a necessity for further experimentation to truly discern the effects of Ihog and/or Boi on the induction of allodynia and hyperalgesia (i.e. is there really alteration of the severity ofÌýinduced allodynia? Or is the increase we see simply proportional to the baseline increase in thermal sensitivity? And can further recordings at hyperalgesic temperatures tease out the differences that seem apparent in the distributions?). Fortunately, there is clear evidence that the exclusive absence of Ihog effects a greater increase in thermal sensitivity. This is a completely new discovery and suggests the possibility that Ihog participates in a signalling pathway distinct from the canonical Hh pathway (since its absence produces hypersensitization). The discovery of such a pathway could describe a whole new range of targets that may be viable to treat allodynia and hyperalgesia in chronic pain sufferers.