Marna E. Ericson

Pain-related behaviors and neurochemical alterations in mice expressing sickle hemoglobin: modulation by cannabinoids

Sickle cell disease causes severe pain. We examined pain-related behaviors, correlative neurochemical changes, and analgesic effects of morphine and cannabinoids in transgenic mice expressing human sickle hemoglobin (HbS). Paw withdrawal threshold and withdrawal latency (to mechanical and thermal stimuli, respectively) and grip force were lower in homozygous and hemizygous Berkley mice (BERK and hBERK1, respectively) compared with control mice expressing human hemoglobin A (HbA-BERK), indicating deep/musculoskeletal and cutaneous hyperalgesia. Peripheral nerves and blood vessels were structurally altered in BERK and hBERK1 skin, with decreased expression of mu opioid receptor and increased calcitonin gene-related peptide and substance P immunoreactivity. Activators of neuropathic and inflammatory pain (p38 mitogen-activated protein kinase, STAT3, and mitogen-activated protein kinase/extracellular signal-regulated kinase) showed increased phosphorylation, with accompanying increase in COX-2, interleukin-6, and Toll-like receptor 4 in the spinal cord of hBERK1 compared with HbA-BERK. These neurochemical changes in the periphery and spinal cord may contribute to hyperalgesia in mice expressing HbS. In BERK and hBERK1, hyperalgesia was markedly attenuated by morphine and cannabinoid receptor agonist CP 55940. We show that mice expressing HbS exhibit characteristics of pain observed in sickle cell disease patients, and neurochemical changes suggestive of nociceptor and glial activation. Importantly, cannabinoids attenuate pain in mice expressing HbS.

Cell death after cartilage impact occurs around matrix cracks.

The damage from rapid high energy impacts to cartilage may contribute to the development of osteoarthritis (OA). Understanding how and when cells are damaged during and after the impact may provide insight into how these lesions progress. Mature bovine articular cartilage on the intact patella was impacted with a flat impacter to 53 MPa in 250 ms. Cell viability was determined by culturing the cartilage with nitroblue tetrazolium for 18 h or for 4 days in medium containing 5% serum before labeling (5‐day sample) and compared to adjacent, non‐impacted tissue as viable cells per area. There was a decrease in viable cell density only in specimens with macroscopic cracks and the loss was localized primarily near matrix cracks, which were in the upper 25% of the tissue. This was confirmed using confocal microscopy with a fluorescent live/dead assay, using 5′‐chloromethylfluorescein diacetate and propidium iodide. Cell viability in the impacted regions distant from visible cracks was no different than the non‐impacted control. At 5 days, viable cell density decreased in the surface layer in both the control and impacted tissue, but there was no additional impact‐related change. In summary, cell death after the impaction of cartilage on bone occurred around impact induced cracks, but not in impacted areas without cracks. If true in vivo, early stabilization of the damaged area may prevent late sequelae that lead to OA.

Mast cell activation contributes to sickle cell pathobiology and pain in mice

Sickle cell anemia (SCA) is an inherited disorder associated with severe lifelong pain and significant morbidity. The mechanisms of pain in SCA remain poorly understood. We show that mast cell activation/degranulation contributes to sickle pain pathophysiology by promoting neurogenic inflammation and nociceptor activation via the release of substance P in the skin and dorsal root ganglion. Mast cell inhibition with imatinib ameliorated cytokine release from skin biopsies and led to a correlative decrease in granulocyte-macrophage colony-stimulating factor and white blood cells in transgenic sickle mice. Targeting mast cells by genetic mutation or pharmacologic inhibition with imatinib ameliorates tonic hyperalgesia and prevents hypoxia/reoxygenation-induced hyperalgesia in sickle mice. Pretreatment with the mast cell stabilizer cromolyn sodium improved analgesia following low doses of morphine that were otherwise ineffective. Mast cell activation therefore underlies sickle pathophysiology leading to inflammation, vascular dysfunction, pain, and requirement for high doses of morphine. Pharmacological targeting of mast cells with imatinib may be a suitable approach to address pain and perhaps treat SCA.

The interactions of Anaplasma phagocytophilum, endothelial cells, and human neutrophils.

Abstract: Ixodes scapularis ticks transmit Anaplasma phagocytophilum (Ap), agent of human granulocytic anaplasmosis (HGA). Invasion of neutrophil granulocytes (PMN) by Ap is the hallmark of the disease, but these short‐lived phagocytes are not likely the sole cell type required for productive infection. We analyzed infection of microvascular endothelial cells during pathogenesis of anaplasmosis in vivo and in vitro. Organs from Ap‐infected mice were processed for confocal microscopy 41 days p.i. Fluorescent labeling of heart and liver sections using anti‐factor VIII and anti‐MSP2 antibodies allowed colocalization of Ap and vascular endothelium, indicating infection. Ap rapidly invaded and grew within HMEC‐1 human microvascular endothelial cells and readily transferred to PMN. Over 50% of PMN became infected within two hours of coincubation with HMEC‐1. PMN adhered to, polarized, and migrated upon infected endothelial monolayers. The Ap receptor on human PMN is PSGL‐1, and infected endothelial cells upregulate ICAM‐1 (CD54), but the mechanisms of transfer of Ap remain unknown. To elucidate the cellular determinants involved, we tested relevant antibodies and lectins. Anti‐PSGL‐1 reduced infection of PMN, but did not inhibit adherence of PMN to Ap infected HMEC‐1 cells while anti‐CD18 did. Sialidase pretreatment increased, and EDTA and fucoidan decreased binding of Ap to HMEC‐1, whereas several other lectins had no effect. An endothelial reservoir of Ap offers opportunities for ongoing, direct cell‐to‐cell infection of PMN, avoidance of host immune effectors, and completion of the Ap life cycle by infection of circulating leukocytes available for transfer to blood‐feeding ticks.

Transient Receptor Potential Type Vanilloid 1 Suppresses Skin Carcinogenesis

Blockade of the transient receptor potential channel vanilloid subfamily 1 (TRPV1) is suggested as a therapeutic approach to pain relief. However, TRPV1 is a widely expressed protein whose function might be critical in various nonneuronal physiologic conditions. The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is overexpressed in many human epithelial cancers and is a potential target for anticancer drugs. Here, we show that TRPV1 interacts with EGFR, leading to EGFR degradation. Notably, the absence of TRPV1 in mice results in a striking increase in skin carcinogenesis. The TRPV1 is the first membrane receptor shown to have a tumor-suppressing effect associated with the down-regulation of another membrane receptor. The data suggest that, although a great deal of interest has focused on TRPV1 as a target for pain relief, the chronic blockade of this pain receptor might increase the risk for cancer development.

Tumor-induced mechanical hyperalgesia involves CGRP receptors and altered innervation and vascularization of DsRed2 fluorescent hindpaw tumors.

Functional and anatomical relationships among primary afferent fibers, blood vessels, and cancers are poorly understood. However, recent evidence suggests that physical and biochemical interactions between these peripheral components are important to both tumor biology and cancer‐associated pain. To determine the role of these peripheral components in a mouse model of cancer pain, we quantified the change in nerve and blood vessel density within a fibrosarcoma tumor mass using stereological analysis of serial confocal optical sections of immunostained hind paw. To this end we introduced the Discoma coral‐derived red fluorescent protein (DsRed2) into the NCTC 2472 fibrosarcoma line using the Sleeping Beauty transposon methodology, thus providing a unique opportunity to visualize tumor–nerve–vessel associations in context with behavioral assessment of tumor‐associated hyperalgesia. Tumors from hyperalgesic mice are more densely innervated with calcitonin gene related peptide (CGRP)‐immunoreactive nerve fibers and less densely vascularized than tumors from non‐hyperalgesic mice. As hyperalgesia increased from Day 5 to 12 post‐implantation, the density of protein gene product 9.5 (PGP9.5)‐immunoreactive nerves and CD31‐immunoreactive blood vessels in tumors decreased, whereas CGRP‐immunoreactive nerve density remained unchanged. Importantly, intra‐tumor injection of a CGRP1 receptor antagonist (CGRP 8–37) partially blocked the tumor‐associated mechanical hyperalgesia, indicating that local production of CGRP may contribute to tumor‐induced nociception through a receptor‐mediated process. The results describe for the first time the interaction among sensory nerves, blood vessels and tumor cells in otherwise healthy tissue, and our assessment supports the hypothesis that direct tumor cell‐axon communication may underlie, at least in part, the occurrence of cancer pain.

Relationship between follicular nerve supply and alopecia

The emergence of new technologies such as the combination of immunohistochemical techniques with laser scanning confocal microscopy allows one to observe and project the three-dimensional perifollicular innervation in tissue sections measuring up to 200 microns. This technology opens the door to making new discoveries about the innervation of the hair follicle. As new information is generated about the cutaneous sensory nervous system, neuropeptide expression, and the modulation of inflammatory and proliferative processes by the nervous system in the skin, it is likely this knowledge will be applied to enhance our understanding of the biology of the hair follicle in both the normal and diseased state.

Intranasal delivery of insulin via the olfactory nerve pathway.

Objectives  Intranasal delivery has been shown to target peptide therapeutics to the central nervous system (CNS) of animal models and induce specific neurological responses. In an investigation into the pathways by which intranasal administration delivers insulin to the CNS, this study has focused on the direct delivery of insulin from the olfactory mucosa to the olfactory bulbs via the olfactory nerve pathway.

A Novel System for Simultaneous in Vivo Tracking and Biological Assessment of Leukemia Cells and ex Vivo Generated Leukemia-Reactive Cytotoxic T Cells

To determine the mechanisms by which adoptive immunotherapy could reduce lethality to acute myelogenous leukemia (AML), a novel technique was developed to track both leukemic blasts and adoptively transferred cytotoxic T cells (CTLs) independently and simultaneously in mice. To follow the fate of ex vivo generated anti-AML-reactive CTLs, splenocytes obtained from enhanced green fluorescent protein transgenic mice were cocultured with AML lysate-pulsed dendritic cells, which subsequently were expanded by exposure to anti-CD3/CD28 monoclonal antibody-coated magnetic microspheres. To track AML cells, stable transfectants of C1498 expressing DsRed2, a red fluorescent protein, were generated. Three factors related to CTLs correlated with disease-free survival: (a) CTL l-selectin expression. l-Selectin high fractions resulted in 70% disease-free survival, whereas l-selectin low-expressing CTLs resulted in only 30% disease-free survival. (b) Duration of ex vivo expansion (9 versus 16 days). Short-term expanded CTLs could be found at high frequency in lymphoid organs for longer than 4 weeks after transfer, whereas long-term expanded CTLs were cleared from the system after 2 weeks. Duration of expansion correlated inversely with l-selectin expression. (c) CTL dose. A higher dose (40 versus 5 × 106) resulted in superior disease-free survival. This survival advantage was achieved with short-term expanded CTLs only. The site of treatment failure was mainly the central nervous system where no CTLs could be identified at AML sites.

Analysis of nociception, sex and peripheral nerve innervation in the TMEV animal model of multiple sclerosis

&NA; Although pain was previously not considered an important element of multiple sclerosis (MS), recent evidence indicates that over 50% of MS patients suffer from chronic pain. In the present study, we utilized the Theiler’s murine encephalomyelitis virus (TMEV) model of MS to examine whether changes in nociception occur during disease progression and to investigate whether sex influences the development of nociception or disease‐associated neurological symptoms. Using the rotarod assay, TMEV infected male mice displayed increased neurological deficits when compared to TMEV infected female mice, which mimics what is observed in human MS. While both male and female TMEV infected mice exhibited thermal hyperalgesia and mechanical allodynia, female mice developed mechanical allodynia at a faster rate and displayed significantly more mechanical allodynia than male mice. Since neuropathic symptoms have been described in MS patients, we quantified sensory nerve fibers in the epidermis of TMEV‐infected and non‐infected mice to determine if there were alterations in epidermal nerve density. There was a significantly higher density of PGP9.5 and CGRP‐immunoreactive axons in the epidermis of TMEV‐infected mice versus controls. Collectively these results indicate that the TMEV model is well suited to study the mechanisms of MS‐induced nociception and suggest that alterations in peripheral nerve innervation may contribute to MS pain.