Faculty Bibliography

Objectives.- To develop a translational mouse model for the study and measurement of non-evoked pain in the orofacial region by establishing markers of nociceptive-specific grooming behaviors in the mouse. Background.- Some of the most prevalent and debilitating conditions involve pain in the trigeminal distribution. Although there are current therapies for these pain conditions, for many patients, they are far from optimal. Understanding the pathophysiology of pain disorders arising from structures innervated by the trigeminal nerve is still limited, and most animal behavioral models focus on the measurement of evoked pain. In patients, spontaneous (non-evoked) pain responses provide a more accurate representation of the pain experience than do responses that are evoked by an artificial stimulus. Therefore, the development of animal models that measure spontaneous nociceptive behaviors may provide a significant translational tool for a better understanding of pain neurobiology. Methods.- C57BL/6 mice received either an injection of 0.9% saline solution or complete Freund's adjuvant into the right masseter muscle. Animals were video-recorded and then analyzed by an observer blind to the experiment group. The duration of different facial grooming patterns performed in the area of injection were measured. After 2 hours, mice were euthanized and perfused, and the brainstem was removed. Fos protein expression in the trigeminal nucleus caudalis was quantified using immunohistochemistry to investigate nociceptive-specific neuronal activation. A separate group of animals was treated with morphine sulfate to determine the nociceptive-specific nature of their behaviors. Results.- We characterized and quantified 3 distinct patterns of acute grooming behaviors: forepaw rubbing, lower lip skin/cheek rubbing against enclosure floor, and hindpaw scratching. These behaviors occurred with a reproducible frequency and time course, and were inhibited by the analgesic morphine. Complete Freund's adjuvant-injected animals also showed Fos labeling consistent with neuronal activation in nociceptive-specific pathways of the trigeminal nucleus after 2 hours. Conclusions.- These behaviors and their correlated cellular responses represent a model of trigeminal pain that can be used to better understand basic mechanisms of orofacial pain and identify new therapeutic approaches to this common and challenging condition.

Our recent findings have suggested an important role for the conditioning of Natural Killer cell effector function by the cancer stem cells as well as healthy untransformed stem cells, stromal monocytes and fibroblasts in cellular differentiation and tissue regeneration. We have also reported that de-differentiation or reversion of tumor cells or healthy non-transformed cells to a less-differentiated stage activated cytotoxic function of NK cells. In this report we examined the function of allogeneic CD8+ T cell cytotoxic function against de-differentiated tumors to determine whether induction of cytotoxic function by de-differentiated tumors is unique to the function of NK cells or that the cytotoxic function of CD8+ T cells is similarly induced when cultured with NFkappaB knock down tumors. Here, we demonstrate that dedifferentiation of tumors by the inhibition of NFkappaB nuclear function sensitizes the tumors to allogeneic CD8+ T cell mediated cytotoxicity, and increased survival and proliferation of T cells. Moreover, increased secretion of IFN-gamma and GM-CSF by CD8+ T cells was observed when these cells were co-incubated with NFkappaB knock down tumors. More importantly, the levels of IL-6 secretion were significantly reduced in the co-cultures of CD8+ T cells and NFkappaB knock down tumors when compared to those obtained from the co-cultures of CD8+ T cells with vector-alone transfected tumors. In addition, treatment of tumor transfectants with IFN-gamma resulted in a decrease in the cytotoxicity and cytokine secretion by CD8+ T cells. However, the function of cytotoxic T cells remained significantly higher in the presence of IFN-gamma treated NFkappaB knock down tumors when compared to either untreated or IFN-gamma treated vector alone transfected tumors. Thus, these results indicated that inhibition of NFkappaB function in tumors activates both NK and CTL functions, suggesting a potential role for both innate and adaptive immune effectors in differentiation and regeneration of the tissues

In recent years bench-based studies have greatly enhanced our understanding of headache pathophysiology, while facilitating the development of new headache medicines. At present, established animal models of headache utilize activation of pain-producing cranial structures, which for a complex syndrome, such as migraine, leaves many dimensions of the syndrome unstudied. The focus on modeling the central nociceptive mechanisms and the complexity of sensory phenomena that accompany migraine may offer new approaches for the development of new therapeutics. Given the complexity of the primary headaches, multiple approaches and techniques need to be employed. As an example, recently a model for trigeminal autonomic cephalalgias has been tested successfully, while by contrast, a satisfactory model of tension-type headache has been elusive. Moreover, although useful in many regards, migraine models are yet to provide a more complete picture of the disorder.

Neurons in sensory ganglia are surrounded by satellite glial cells (SGCs) that perform similar functions to the glia found in the CNS. When primary sensory neurons are injured, the surrounding SGCs undergo characteristic changes. There is good evidence that the SGCs are not just bystanders to the injury but play an active role in the initiation and maintenance of neuronal changes that underlie neuropathic pain. In this article the authors review the literature on the relationship between SGCs and nociception and present evidence that changes in SGC potassium ion buffering capacity and glutamate recycling can lead to neuropathic pain-like behavior in animal models. The role that SGCs play in the immune responses to injury is also considered. We propose the term gliopathic pain to describe those conditions in which central or peripheral glia are thought to be the principal generators of principal pain generators.

Freshly isolated untreated NK cells undergo rapid apoptosis and lose their cytotoxic function upon the addition of F(ab')2 fragment of anti-CD16 antibodies. Loss of NK cell cytotoxic function after treatment with F(ab')2 fragment of anti-CD16 antibody can be seen against K562 and UCLA-2 oral tumor cells when either added immediately in the co-cultures of NK cells with the tumor cells or after pre-treatment of NK cells with the antibody before their addition to the tumor cells. Addition of Interleukin-2 (IL-2) in combination with anti-CD16 antibody to NK cells delayed the induction of DNA fragmentation in NK cells, and even though decreased cytotoxicity could still be observed against K562 and UCLA-2 oral tumors when compared to IL-2 alone treated NK cells, the cytotoxicity levels remained relatively higher and approached those obtained by untreated NK cells in the absence of antibody treatment. No increases in IFN-gamma, Granzymes A and B, Perforin and TRAIL genes could be seen in NK cells treated with anti-CD16 antibody. Neither secretion of IFN-gamma nor increased expression of CD69 activation antigen could be observed after the treatment of NK cells with anti-CD16 antibody. Furthermore, IL-2 mediated increase in CD69 surface antigens was down-modulated by anti-CD16 antibody. Finally, the addition of anti-CD16 antibody to co-cultures of NK cells with tumor target cells was not inhibitory for the secretion of VEGF by oral tumor cells, unlike those co-cultured with untreated or IL-2 treated NK cells. Thus, binding and triggering of CD16 receptor on NK cells may enhance oral tumor survival and growth by decreased ability of NK cells to suppress VEGF secretion or induce tumor cell death during the interaction of NK cells with oral tumor cells.

The aim of this study is to identify the phenotype of resistant oral tumors, and to delineate the contribution of immune effectors to resistance of oral tumors. UCLA-1 oral tumors which were resistant to NK cell mediated cytotoxicity secreted increased amounts of IL-6, IL-1beta, GM-CSF, and IL-8 when cultured with or without immune effectors. In addition, the levels of vascular endothelial growth factor (VEGF) secretion in the co-cultures of naive immune effectors with UCLA-1 rose significantly when compared to tumor cells alone. IL-2 activated NK cells decreased VEGF secretion in all tumor cells. However, NK cells which were induced to undergo cell death with anti-CD16 antibody were not only unable to decrease VEGF secretion, but they also contributed further to the increase in VEGF secretion by oral tumors. Overall, we show in this paper that naive as well as non-viable immune effectors may contribute to the growth and resistance of oral tumors by triggering the secretion of key tumor cell growth factors.

The inhibitory role of TNF-alpha on survival of naive and IL-2 treated NK cells has been demonstrated in the past. However, its effect on the function of these cells against tumor cells, in particular against oral tumors has not been established. We investigated the significance of secreted TNF-alpha in death and functional loss of splenocytes and NK cells in ex-vivo cultures with oral tumors. Oral tumors trigger potent secretion of TNF-alpha by human and murine immune effectors. Absence of TNF-alpha increases the cytotoxic activity and secretion of IFN-gamma by IL-2 treated splenocytes and NK cells in co-cultures with MOK L2D1+/p53-/- oral tumor cells. IL-2 treated splenocytes and NK cells from TNF-alpha -/- mice survive and proliferate more when compared to cells from TNF-alpha +/+ mice. Cell death induced by F. nucleatum, an oral bacteria, in TNF-alpha -/- splenocytes are considerably lower than that induced in TNF-alpha +/+ splenocytes where potent release of TNF-alpha is reproducibly observed. Addition of exogenous rTNF-alpha to IL-2 treated splenocytes and NK cells decreased survival and function of splenocytes and NK cells obtained from TNF-alpha -/- mice against oral tumors. These findings suggest that potent induction of TNF-alpha during interaction of immune effectors with oral tumors and/or oral bacteria is an important factor in decreasing the function and survival of cytotoxic immune effectors. Strategies to neutralize TNF-alpha may be beneficial in the treatment of oral cancers.

Currently the clinical needs for pain and headache management are not met. Despite the numerous and exciting recent advances in understanding the molecular and cellular mechanisms that originate pain, we cannot yet fully explain the mechanism underlying the biology of chronic pain. Pain is a natural mechanism preserving our species survival; however, when the protective quality is lost, physiologic changes to the peripheral and central nervous systems result in the formation of chronic pain states. Once we understand how this chronic pain state is created, either through genetic, environmental, therapeutic, or other triggers we may be able to enhance our species existence, limiting maladaptive pain and suffering. The future therapeutic targets will need to address the genetics, neurophysiologic changes of the neurons and brain as well as help control immune systems including the glia. The key to successful headache and pain therapy is research aimed at prevention and minimizing the plastic changes triggering chronic pain.

The objective was to retrospectively characterise the efficacy of memantine as preventive therapy in a series of patients with frequent migraine. Patients in a university headache clinic completed a survey regarding their experience with memantine, and medical records were reviewed. All patients who received memantine as preventive therapy for migraine over a 15-month period were mailed surveys and consent forms for record review. Patients were treated with memantine beginning at a dose of 5 mg/day, increasing if needed by 5 mg/week up to 10 mg twice a day. The majority of patients (36 out of 54) treated with memantine for at least 2 months reported a significant reduction in estimated headache frequency, and improved function. Side effects were uncommon and generally mild. This limited retrospective case review suggests that memantine may be an effective preventive therapy for patients with frequent migraine. A prospective trial is warranted.

The prevalence of migraine is much greater in female than male individuals. Cortical spreading depression (CSD) is thought to be a fundamental mechanism of migraine, and CSD in rodents is used as a model for migraine. We used optical intrinsic signal imaging and electrophysiological techniques to investigate CSD in C57Bl/6 mice. Using two different methods for induction of CSD, we found that female mice had a significantly reduced threshold for induction of CSD compared with male mice. These results suggest an increased cortical excitability in female mice that may be independent of the estrous cycle.