Michael P. Jankowski, PhD - US grants

ABSTRACT: Musculoskeletal pain resulting from tissue ischemia with reperfusion is a major health issue that affects millions of people in the United States. Peripheral ischemia/ reperfusion occurs in blood disorders like sickle cell disease, and in cardiovascular disorders such as peripheral vascular disease. Ischemia/ reperfusion is also thought to be the underlying cause of complex regional pain syndrome. While much is known about the functional properties and plasticity of cutaneous nociceptors following peripheral injuries and how these fibers contribute to pain, relatively little is known about the functional properties of group III and IV muscle afferents and their role in muscle pain development. The major goal of this proposal is to determine the molecular mechanisms of muscle afferent sensitization that may underlie muscle pain during ischemia and after tissue reperfusion. We hypothesize that these distinct phases cause differential changes in heat, mechanical and chemo-sensitivity in muscle afferents, which are mediated by upregulation of purinergic receptors during ischemia and acid sensing ion channels after reperfusion leading to muscle pain. In order to increase our knowledge of muscle afferents, we developed a novel ex vivo forepaw muscle, median & ulnar nerves, dorsal root ganglion (DRG), spinal cord recording preparation that enables us to comprehensively phenotype these afferents in mouse. We are also able to analyze the central anatomy, and the neurochemical or molecular phenotypes of these afferents using combinations of ex vivo recording with immunocytochemical and single cell RT-PCR analyses. In Specific Aim 1, we will determine if upregulation of purinergic receptors, P2Y1 and P2X5, regulate the observed changes in heat and chemosensitivity in muscle afferents, respectively during ischemia using in vivo siRNA-mediated knockdown of these genes in single peripheral nerves in conjunction with ex vivo recording preparations. Next, in Specific Aim 2, we will utilize a similar approach to SA1 except we will determine if upregulation of ASIC1 and ASIC3 regulate the novel changes in mechanical and metabolite responses in muscle afferents, respectively after transient ischemia with tissue reperfusion. Finally, in Specific Aim 3, we will determine if upregulation of P2Y1 and P2X5 regulate muscle pain during ischemia while upregulation of ASIC1 and ASIC3 regulate muscle pain after reperfusion by analyzing the effects of receptor knockdown on recognized muscle pain behavior tests between these two phases. This study will enable us to characterize the changes in both non-nociceptive and nociceptive muscle afferents after ischemia/ reperfusion and identify unique mechanisms associated with muscle afferent sensitization that underlie muscle pain development. This may lead to the formulation of more appropriate treatments for musculoskeletal pain associated with ischemia/ reperfusion that target the proper pain receptor(s) or primary afferent subpopulation(s).

DESCRIPTION (provided by applicant): Numerous pediatric disorders and surgical procedures performed on children lead to pain states yet it is well known that current pharmacological treatments for pain have adverse effects in children such as mild nausea or vomiting but also more severe side effects like respiratory depression and even death. Thus in order to develop more appropriate therapies for pain in children, a better understanding of how particular sensory afferent populations may contribute to pediatric pain states is of utmost importance. While much is known about the functional properties and plasticity of cutaneous nociceptors following peripheral injuries in adults, relatively little is known about the functiona properties of sensory afferents during development and the mechanisms of sensitization after peripheral injury. The main goal of this proposal is to functionally characterize cutaneous sensory afferents throughout development in naive mice and in models of postnatal inflammation, and begin to determine some of the mechanisms involved with the changes in sensory afferents. In order to determine this, we developed an ex vivo hairy skin, saphenous nerve, dorsal root ganglion (DRG), spinal cord recording preparation in neonatal/ postnatal mice that enables us to comprehensively phenotype sensory fibers before and after cutaneous injury. In Specific Aim 1, we will first analyze the functional, anatomical, and neurochemical phenotypes of these afferents at various times during postnatal development using combinations of ex vivo recording and immunocytochemical analyses. Changes in function will then be correlated with alterations in mRNA and protein expression of various receptors/ channels thought to be involved in sensory function in the DRGs. Next, in Specific Aim 2, we will perform similar studies as described in SA1 except we will analyze the comprehensive phenotypes of sensory afferents after hairy skin injection of carrageenan at different times during postnatal development and correlate these data with changes in gene expression in the DRGs to determine the potential mechanisms of the observed functional changes in cutaneous afferents. These experiments will enable us to characterize the changes in all types of cutaneous sensory neurons throughout development and during peripheral inflammation, and potentially identify unique age-dependent mechanisms associated with how developing sensory neurons respond to injury. These studies will establish the foundation of future experimentation using in vivo siRNA- mediated knockdown of genes in single peripheral nerves in conjunction with ex vivo recording preparations where we will be able to analyze the roles of changes in specific receptors/ channels during normal (uninjured) development and during different times of postnatal cutaneous inflammation. These and future studies will allow us to better understand how changes in sensory fibers impact pediatric pain and may also lead to the establishment of more suitable treatments for pain in children.

ABSTRACT: Numerous pediatric disorders and surgical procedures performed on children lead to pain states yet it is well known that current pharmacological treatments for pain have significant adverse effects in children. Thus in order to develop more appropriate therapies for pediatric pain, a better understanding of how pain states are generated in children is of utmost importance. However, compared to adults, we know relatively little about the mechanisms of pediatric pain development. Patients with growth hormone deficiency (GHD) however, may provide insight into this clinical problem since these patients often display pain at rest in addition to deficits in growth. Moreover, treatment of these pain patients with GH provides analgesia. We have also found in neonatal mice with GHD that they display hyper-responsiveness to mechanical and thermal stimuli and the sensory afferents in these mutants are also sensitized to these stimuli. In addition, we found that cutaneous inflammation in normal mice produces a GHD state in the injured skin and observed sensitization of primary afferents and changes in pain-related behaviors during inflammation were blocked by GH treatment via inhibition of insulin like growth factor 1 receptor (IGFr1) upregulation in the DRGs. Finally, a neonatal GH treatment was also effective in blocking the priming effects of early life insults on young adult hypersensitivity. The main goal of this proposal is to determine the molecular mechanisms of how GH levels regulate sensory neuron development and afferent sensitization during inflammation that may underlie short and long term mechanical and thermal responsiveness. To determine this, we developed an ex vivo hairy skin, saphenous nerve, dorsal root ganglion (DRG), spinal cord recording preparation in neonatal mice that enables us to comprehensively phenotype sensory fibers. In Specific Aim 1, we will determine how GH modulates sensory neuron development by using transgenic mice with GHD or mice with afferent specific GH receptor ablation in conjunction with ex vivo recording. In Specific Aim 2, we will test whether knockdown of the receptor (IGFr1) or transcription factor (SRF) that is thought to mediate the actions of altered GH levels modifies these same changes in afferent function in GHD mice using in vivo siRNA-mediated knockdown of these genes in single peripheral nerves in conjunction with ex vivo recording. Finally, in Specific Aim 3, we will use ex vivo recording to determine the influence of treating neonatally inflamed mice with GH on the priming effects to subsequent adult inflammation. All of these aims will be complemented by behavioral analysis of thermal and mechanical responsiveness. These experiments will enable us to determine if GH levels regulate sensory development and identify unique mechanisms associated with how developing sensory neurons respond to injury. These studies will also facilitate our understanding of the transition from acute to chronic pediatric pain, and will allow us to determine the utility of GH as a pain therapy. This work may also lead to the establishment of more suitable treatments for pain in children that target the correct pain receptor(s) or primary afferent subtype(s).

ABSTRACT: Approximately 15-20% of children experience persistent or chronic pain. However, compared to adults, we know relatively little about the mechanisms of pediatric pain development. A basic understanding of nociceptive processing in the immature nervous system is therefore crucial in order to develop more appropriate treatments for pain in children. Patients with growth hormone deficiency (GHD) may provide insight into this clinical problem. Patients with GHD often display pain at rest in addition to deficits in growth. Moreover, treatment of certain pain patients with GH provides analgesia. We have found that cutaneous inflammation and muscle incision in mice reduces GH in the injured tissues. Observed changes in gene expression, afferent function and pain-related behaviors during neonatal injury are blocked by treating mice with exogenous GH. New pilot data suggests that macrophage dependent sequestering of GH at the site of peripheral injury, subsequently reduces inhibitory microRNA expression (e.g. miR-133a) within nociceptors to increase transcription factor (e.g. serum response factor (SRF)) dependent upregulation of various receptors and channels that modulate afferent function and pain-related beahviors. The main goal of this proposal is to determine the molecular mechanisms of how GH levels regulate sensory neuron sensitization during muscle incision and how this may underlie acute and persistent neonatal hypersensitivity. Specific Aim 1 will use a novel ex vivo somatosensory recording preparations to determine the effects of macrophage or sensory neuron specific knockout of the GH receptor on the sensitization of sensory neurons in uninjured neonatal mice or animals with muscle incision. Specific Aim 2 will test whether knockdown of a transcription factor (SRF) or overexpression of a microRNA (miR-133a), that is thought to regulate receptor expression in neurons (and thereby modulate peripheral sensitization), modifies these same changes in afferent function after muscle incision using in vivo siRNA-mediated knockdown or plasmid based overexpression strategies in single peripheral nerves in conjunction with ex vivo recording. Each of these two aims will be complemented by analysis of ongoing and evoked hypersensitivity. Finally, Specific Aim 3 will use behavioral analyses and/or ex vivo recording to determine the influence of localized GH treatments, GHr KO, SRF inhibition or miR-133a overexpression in neonatally incised mice on the prolonged effects to subsequent adolescent incision. These experiments will allow a better understanding of the unique mechanisms in primary sensory neurons by which peripheral GH levels regulate afferent sensitization and neonatal pain development. These studies will facilitate understanding of the transition from acute to chronic pediatric pain, and will allow us to determine the utility of GH as a pain therapy for children. This work may also lead to the design of more suitable treatments for pediatric pain that target specific pain receptor(s), signaling molecule(s) or afferent subtype(s).

Abstract: Peripheral injury responses require sophisticated interactions of target tissues, immune cells and primary sensory neurons. Crosstalk between these systems is essential for post-injury muscle repair and nociception. While a great deal is known about the role of the immune system in functional restoration of muscles and in pain development, it is not known if physical coupling of circulating immune cells to myofibers or neurons after injury directly modulates both of these unique biological processes. Information on novel interactions between muscles, the peripheral nervous system and immune cells could significantly advance understanding of myalgia and muscle repair. The goal of this study is to determine if infiltrating immune cells electrically couple to myofibers or neurons after injury to dually modulate functional muscle repair and nociception. Recent reports suggest that, after injury, connexin 43 (Cx43) gap junctions may form between macrophages and myofibers to modulate repair. It has also been shown that similar gap junctions form between adjacent neurons within the dorsal root ganglion (DRG) to regulate nociception. In the heart, electrical coupling between resident macrophages and cardiomyocytes is crucial for proper atrioventricular conduction. It is therefore reasonable to hypothesize that immune cells electrically couple to myofibers and nociceptors after skeletal muscle damage to coordinate responses to injury and dually modulate tissue repair and pain. Aim 1 (R61 Phase) will determine if electrical coupling of macrophages to nociceptors modulates incision-related hypersensitivity. This study will use novel transgenic strategies to specifically knockout Cx43 in macrophages in mice with hind paw muscle incision. This will be used in conjunction with chemogenetic or sono-genetic activation of macrophages. Impact of Cx43 knockout and macrophage activation will be assessed with our ex vivo muscle afferent recording preparations, muscle pain-related behavioral tests and calcium imaging (using GCaMP6 reporters) in co-cultures of macrophages and primary DRG cells. Aim 2 (R61 Phase) will use similar groups to determine if electrical coupling of infiltrating macrophages to myofibers facilitates repair of muscle tissue after incision. The impact of these manipulations will be determined using calcium imaging of hind paw muscle cells, electromyography/ compound muscle action potential recordings in vivo, and anatomical analyses of muscle membrane integrity. Aim 3 (R33 Phase) will further explore the functions of macrophage electrical coupling using different transgenic combinations, inhibitory chemogenetics and more severe models of muscle injury. Results will allow determination of novel means of communication between circulating immune factors and the peripheral structures they are affecting. Data will provide novel insights into muscle injury responses that will go well beyond the incremental expansion of current reports. These insights could identify a novel target for therapeutic intervention for pain or muscle repair in numerous muscle injury states.