Erin D. Milligan, Ph.D. - US grants

DESCRIPTION (provided by applicant): The present proposal is an extension of an ongoing 2-yr R21 (Watkins, P.I.) under the NIDA CEBRA program. Its aims are focused on developing a new therapy for pain. Controlling chronic pain in humans is a major unresolved problem. Recent data strongly suggest that spinal cord gila (astrocytes & microglia) are critically involved in the creation & maintenance of diverse enhanced pain states. Spinal cord gila create enhanced pain via the release of proinflammatory cytokines (PlCs): tumor necrosis factor (TNF), interleukin-1 (IL1) & interleukin-6 (IL6). Recognition of the key importance of spinal cord gila & glial PICs in pathological pain opens new avenues for pain control. There are various pharmacological means available to control glial dysregulation of pain. Interleukin-10 (IL10) is very promising from a clinical point of view.' IL10 is an anti-inflammatory cytokine, which acts as an endogenous suppressor of proinflammatory cytokine production & activity. IL10 is an excellent candidate for preventing & reversing PIC-driven pathological pain states. However, two practical problems need to be overcome. First, control of chronic pain requires chronic delivery of IL10. Second, IL10 cannot cross the blood-brain barrier, thus negating systemic administration. To resolve these issues, we are exploring the feasibility of prolonged spinal release of Ll10 induced by gene therapy. Here, vectors encoding IL10 are injected into the cerebrospinal fluid surrounding the spinal cord (intrathecal; IT), so as to mimic a clinically relevant route of delivery. Our preliminary data provide strong support that spinal gene therapy with IL10 will prevent & reverse enhanced pain states. The aims of the present proposal are straightforward: (1) To identify the optimal vectors from a limited number of candidates, in terms of their effectiveness in transcribing the gene of interest & reversing clinically relevant pain models; (2) To examine the mechanisms by which these optimal IL10-inducing vectors exert their effects in spinal cord; and (3) to examine potential short-comings of this approach. Together, these studies will test the premise that gene therapy with IL10 is worthy of clinical development for controlling diverse pathological pain states. This approach to pain control represents a dramatic departure from all other available therapies.

DESCRIPTION (provided by applicant): The present proposal is a competing renewal of a Stage II NIDA Cutting Edge Basic Research Award (CEBRA). Its aims are focused on developing a new therapy for persistent pain relief. Persistent pain (3+ months) is a common, unresolved health problem in Americans. A recent consideration in our understanding of neuropathic pain (pathological neuronal signaling in the pain pathway) includes the contribution of immune cells & glia (astrocytes, microglia, Satellite & Schwann) in pain relevant compartments such as the spinal cord dorsal horn, spinal meninges associated subarachnoid matrix, & dorsal root ganglia. Spinal cord glia mediate pathological pain via the release of well-characterized proinflammatory cytokines. The anti-inflammatory cytokine, interleukin-10 (IL-10), potently inhibits proinflammatory cytokine actions. During the current grant period, data strongly support spinal subarachnoid (intrathecal; IT) gene delivery of IL-10 prevents & reverses pathological pain in animal models. Long-duration (3+ months) pain relief is achieved upon 2 sequential IT injections of non-viral vectors, where the 2nd injection must encode IL-10 (plasmid DNA encoding IL-10; pDNA- IL-10). The first injection serves to sensitize the spinal subarachnoid compartment to the 2nd injection that creates IL-10-dependent long-duration pain relief. A robust accumulation of glia, macrophage &/or dendritic cells are components of sensitization. IL-10 protein signaling during the sensitization interval is necessary for long-duration IL-10 gene therapy. We postulate that several immune interrelated etiologies, including chemotaxis, mitosis, & phagocytosis play critical roles for sensitization & IL-10 transgene uptake. Thus, activated (chemotactic &/or mitotic) immune cells & glia could be responsible, in part, for the sensitized response to pDNA-IL-10 uptake. An FDA-approved synthetic polymer improves pDNA-IL-10 drug delivery after a single injection at reduced dosage formulations. However, further improvement is needed for clinical trials. Identifying the anatomical region, cell type and cellular activity underlying sensitization can be exploited to further improve polymer spinal IL-10 targeted gene delivery. The aims of the present proposal are straightforward: (1) To identify the cellular/biochemical responses in pain-relevant regions during sensitization; (2) To examine whether the cellular/biochemical profiles important during sensitization are also necessary during long duration gene expression and pain relief; and (3) To further improve IT gene delivery using PLGA- pDNA-IL-10 formulations that include co-release of factors important during sensitization & long-duration gene expression such that enduring pain relief can be achieved from a single injection.

DESCRIPTION (provided by applicant): Chronic neuropathic pain, such as allodynia, defined as painful sensation to usually non-painful stimuli, is mediated by pathologically activated glial cells residing in both sensory dorsal root ganglia (DRG) and in the spinal cord. These cells secrete the pro-inflammatory factors, IL-1b and TNF-a, as well as chemokines, such as CCL2, that attract circulating leukocytes to the DRG and spinal cord regions. These leukocytes cross the blood-nerve and -spinal barriers, a process facilitated by the activation of the b-2 (b2)-integrin, lymphocyte function-associated antigen-1 (LFA-1), providing an additional source of IL-1b and TNF-a exacerbating neuronal pain signaling processes. Underlying factors that may predispose individuals to allodynia are generally not well recognized. Increasing evidence indicates that prenatal alcohol exposure (PAE) can produce long-lasting alterations to immune function and neuroimmune interactions, increasing the risk for a number of chronic immunologic disorders such as diabetes and rheumatoid arthritis. Specifically, PAE is thought to pathologically prime immune-like glial cells (satellite cells, astrocytes and microglia) disrupting their nutritive role in support of neuronal function during development and into adulthood. Whether PAE leads to heightened and enduring glial reactivity throughout adulthood leading to aberrant neuroimmune signaling is not well understood. The objective of this proposal is to examine whether moderate PAE can heighten allodynic responses in adult offspring using a well-established rodent model of chronic peripheral neuropathic pain, and by applying this model system, to understand underlying PAE-related aberrant neuroimmune interactions. Our overarching hypothesis states that: PAE produces chronic gliopathy and DRG and spinal leukocyte accumulation that mediate pathological pain in rats. The Specific Aims of the proposal are to: (1) identify the magnitude and role of DRG & spinal leukocyte accumulation in the development of neuropathic pain in adult PAE rats, and (2) identify the DRG and spinal cytokine/chemokine, glial activation and b2-integrin expression profile in PAE neuropathic rats. The studies could provide novel insights into whether an adverse in utero environment intersects with vulnerability to developing adult onset allodynia, as well identifying potential neuroimmune makers for discerning vulnerability to neuropathological pain, and more broadly, to disorders with an underlying neuroinflammatory component. These studies could also inform future studies targeting the development of interventional approaches for the amelioration of neuropathic pain in individuals predisposed to this chronic disabling neurologic disorder.

PROJECT SUMMARY Exposure to alcohol during gestation can lead to a constellation of mild to severe disabilities that includes cognitive and behavioral deficits representing a continuum referred to as Fetal Alcohol Spectrum Disorders (FASD), with a prevalence of ~4.8% in some US regions. A growing body of evidence strongly implicates the adverse impact of alcohol exposure during central nervous system (CNS) development on cellular and molecular programing of neuroimmune function. In animal models of prenatal alcohol exposure (PAE), expression of the brain's immune signaling molecules, the proinflammatory cytokines interleukin-1? (IL-1?), tumor necrosis factor-alpha (TNF-?) and the chemokine CCL2, are significantly elevated. While evidence of sensory abnormalities including tactile sensitivity observed in children with FASD are thought to be a result of psychosocial factors, the underlying cause may include neurological dysfunction. Indeed, animal models of PAE reveal heightened sensitivity to light touch, a well-known pathological sensory condition mediated by aberrant neuronal actions in the spinal cord. Clinically, touch hypersensitivity is known as allodynia in chronic pain patients, and animal models of allodynia show pathological activation of pain neurons occurs in the spinal cord mediated by IL-1?, TNF-? and CCL2. Glial cells (astrocytes & microglia) are key producers of these proinflammatory cytokines. Thus, animal models of allodynia and PAE reveal a surprising neuroimmune overlap. Studies of allodynia in animals show peripheral leukocytes traffic to the spinal cord in response to CCL2. Notably, leukocytes cross spinal microvascular endothelial cells (MECs) into the CNS by the action of the ?2-adhesion molecule, lymphocyte function associated antigen 1 (LFA-1), and importantly, glial cells control the healthy barrier function of MECs. Curiously, evidence shows PAE causes structural abnormalities at the glial CNS-MEC interface. Thus, the long-term goal is to identify spinal MEC & neuroimmune adaptations in PAE male and female offspring that enhance adult susceptibility to neuropathy. New therapeutic targets to alleviate aberrant neuroimmune function may be identified. The overall objective will identify the impact PAE exerts on responses of spinal immune adaptations to minor peripheral nerve & immune challenge in males & females. Overarching Hypothesis: PAE potentiates spinal and peripheral proinflammatory immune responses in the nociceptive pathway creating susceptibility for chronic neuropathy from minor insult or challenge. The Aims of the proposal will: (I) Examine the impact of PAE on cytokine profile and function in mediating neuropathy from minor insults and immune challenges in adults, (II) Determine the functional consequences of PAE-induced tight junction defects of the blood-spinal barrier on neuropathy, and (III) Determine PAE-induced defects of the peripheral immune response underlying susceptibility to neuropathy. Results will provide new knowledge for understanding the developmental origins of aberrant PNS- and CNS- immune interactions due to PAE, revealing susceptibilities to adult onset diseases such as neuropathic pain.