Investigations in acute and chronic allodynia : using human psychophysics, sensory analysis, glial modulation & functional proteomics in the dorsal horn of the spinal cord

Abstract

In many chronic pain pathologies, stimuli that are normally non-painful to healthy humans, such as light touch or small decrements in temperature, can cause painful responses – a phenomenon called allodynia. The majority of research on tactile allodynia has focused on myelinated low threshold A mechanoreceptors (A-LTMR) that are otherwise known to mediate innocuous mechano-sensation (1, 2). Recently, there is a growing recognition of the contribution of more than one class of low threshold fibres to pain processing, i.e. unmyelinated low threshold C mechanoreceptors (C-LTMRs), which are believed to be responsible for affective touch processing. (3-6). The first evidence of the contribution of C tactile fibres (CTs), the human counterpart of C-LTMRs, to cutaneous allodynia was shown in human models of rapid-onset skin and muscle pain as well as delayed-onset muscle soreness (3-9). In addition, recent studies in transgenic mice, in both acute and chronic pain models, have provided molecular mechanisms at the spinal level, which functionally correspond to the tactile and cold allodynia perceived in human subjects (10-13). In the last few years, both animal and human studies have argued for a convergence of low threshold C fibre input to nociceptive fibre signalling at the dorsal horn level (3, 4, 7, 10, 14-17). On the basis of the behavioural observations of bilateral cold and tactile allodynia post unilateral median nerve injury (from Paper I) in this thesis, it is hypothesised that central interactions of fibres conducting noxious (i.e. nociceptors) as well as non-noxious stimuli (i.e.non-nociceptors) at the dorsal horn level are responsible in driving these sensations. In addition, the actions of spinal immune cells in the dorsal horn such as microglia and astrocytes to influence nociceptive processing have been firmly established (18-22). Akin to the canonical perspective of attributing allodynia to A-LTMRs, the role of spinal glial cells in injury induced hypersensitivity has always been looked at in the context of these ALTMRs in addition to the unmyelinated high threshold C nociceptors (20, 23, 24). From previous observations of such neuro-glial modulation mentioned above, it is hypothesized that the glial cells can modulate bilateral noxious signalling and the percept of allodynia evoked by otherwise innocuous stimuli, i.e. normally non-painful touch and cooling in this model of bilateral allodynia due to a unilateral nerve injury. Incidentally, rapid cooling and non-painful gentle touch are hallmark of CTs in humans (5, 6) and in mice (10, 12, 13) and hence, cannot be ignored as one of the likely substrates of this phenomena in this rat model. In the dorsal horn, lamina II, which is the main termination site of CT inputs, has been postulated to act as a 'gate' for processing noxious as well as innocuous information by converging and processing inputs from superficial as well as deep dorsal horn (25). This work supports the view, also discussed in Abraira, Kuehn (26), that of low and high threshold inputs from the periphery, it is the dorsal horn (lamina I-III), that acts as the primary convergence centre to 'gate' or 'gain' painful and non-painful information before it is passed over to higher order neurons. Furthermore, by studying the neuronal and nonneuronal interactions in the dorsal horn at the peak time point of the bilateral allodynia seen in our model, it is possible to understand the biochemical changes responsible for this behaviour. These changes help us understand key mechanisms of protein signalling at the cellular dorsal horn that has been often overlooked in other experiments involving proteomics due to experimental bias. Hence, the animal studies in this thesis provide a greater understanding of mechanisms of allodynia and its modulation using pharmacological tools in animals. In rats, following a unilateral median nerve injury, bilateral allodynia was ameliorated using minocycline, a glial inhibitor, by modulation of the dorsal horn interactions between glia and primary afferent fibres that respond to non-painful stimuli. These interactions (Paper I & II) were studied using immuno-staining and proteomic profiling of the dorsal horn – prior to and following nerve injury and its modulation by minocycline. The key result from the animal work, presented in this thesis, is the presentation of modulatory proteins responsible for the sensory behaviour at the contralateral, uninjured side as opposed to the primarily studied injured side of the dorsal horn. To understand the ‘gating’ and ‘gaining’ mechanism of the dorsal horn discussed previously in rodent models, it is important to ask what dissimilates the perception of painfulness from non-painfulness or pleasure in humans with acute pain. Therefore, in healthy humans (Paper III), pain modulation was tested in the context of acute background muscle pain with concurrent ‘affective’ tactile stimulation. Furthermore, the resulting percept and its peripheral substrates was determined using a preferential block of myelinated fibres. In this study, we demonstrated that activation of CTs can produce a stimulus-locked, bi-directional (excitatory/inhibitory) affective modulation of pain that is consistent with previously documented CT function in the rodent dorsal horn. From this human study, it is hypothesised that CT dependent context driven affective stimulus can determine the direction of the perception, in this case, pleasurable or painful. This study also presents with the idea that long term changes in the dorsal horn circuitry may not be necessary to initiate this type of context-dependent bi-directional stimulation and can be achieved in an acute setting. Hence, the animal and human studies combine the animal sensory behaviour and human perception of allodynia (and its modulation) to explore the link between the activation of low threshold sensory fibres such as CTs (by innocuous stimuli) and nociceptive processing in acute and pathological pain states. This thesis comprises of an introduction section and 3 manuscripts. The introduction provides a short review of the peripheral and central mechanisms of allodynia with the aim of interlacing it with the work presented in the 3 manuscripts (referred to in the text by their Roman numerals)