Inflammatory Renin-Angiotensin System Disruption Attenuates Sensory Hyperinnervation and Mechanical Hypersensitivity in a Rat Model of Provoked Vestibulodynia.

Vestibulodynia is characterized by perivaginal mechanical hypersensitivity, hyperinnervation, and abundant inflammatory cells expressing renin-angiotensin system proteins. We developed a tractable rat model of vestibulodynia to further assess the contributions of the renin-angiotensin system. Complete Freund's adjuvant injected into the posterior vestibule induced marked vestibular hypersensitivity throughout a 7-day test period. Numbers of axons immunoreactive for PGP9.5, calcitonin gene-related peptide, and GFRα2 were increased. Numbers of macrophages and T cells were also increased whereas B cells were not. Renin-angiotensin-associated proteins were abundant, with T cells as well as macrophages contributing to increased renin and angiotensinogen. Media conditioned with inflamed vestibular tissue promoted neurite sprouting by rat dorsal root ganglion neurons in vitro, and this was blocked by the angiotensin II receptor type 2 receptor antagonist PD123319 or by an angiotensin II function blocking antibody. Sensory axon sprouting induced by inflamed tissue was dependent on activity of angiotensin-converting enzyme or chymase, but not cathepsin G. Thus, vestibular Complete Freund's adjuvant injection substantially recapitulates changes seen in patients with provoked vestibulodynia, and shows that manipulation of the local inflammatory renin-angiotensin system may be a useful therapeutic strategy. PERSPECTIVE: This study provides evidence that inflammation of the rat vestibule induces a phenotype recapitulating behavioral and cytological features of human vestibulodynia. The model confirms a crucial role of the local inflammatory renin-angiotensin system in hypersensitivity and hyperinnervation. Targeting this system holds promise for developing new nonopioid analgesic treatment strategies.

A Local Inflammatory Renin-Angiotensin System Drives Sensory Axon Sprouting in Provoked Vestibulodynia.

Vestibulodynia is a form of provoked vulvodynia characterized by profound tenderness, hyperinnervation, and frequently inflammation within well-defined areas of the human vestibule. Previous experiments in animal models show that inflammatory hypersensitivity and hyperinnervation occur in concert with establishment of a local renin-angiotensin system (RAS). Moreover, mechanical hypersensitivity and sensory axon sprouting are prevented by blocking effects of angiotensin II on angiotensin II receptor type 2 (AT2) receptors. This case-control study assessed whether a RAS contributes to hyperinnervation observed in human vestibulodynia. Vestibular biopsies from asymptomatic controls or patients' nontender areas showed moderate innervation and small numbers of inflammatory cells. In women with vestibulodynia, tender areas contained increased numbers of mechanoreceptive nociceptor axons, T-cells, macrophages, and B-cells, whereas mast cells were unchanged. RAS proteins were increased because of greater numbers of T cells and B cells expressing angiotensinogen, and increased renin-expressing T cells and macrophages. Chymase, which converts angiotensin I to angiotensin II, was present in constant numbers of mast cells. To determine if tender vestibular tissue generates angiotensin II that promotes axon sprouting, we conditioned culture medium with vestibular tissue. Rat sensory neurons cultured in control-conditioned medium showed normal axon outgrowth, whereas those in tender tissue-conditioned medium showed enhanced sprouting that was prevented by adding an AT2 antagonist or angiotensin II neutralizing antibody. Hypersensitivity in provoked vestibulodynia is therefore characterized by abnormal mechanonociceptor axon proliferation, which is attributable to inflammatory cell-derived angiotensin II (or a closely related peptide) acting on neuronal AT2 receptors. Accordingly, reducing inflammation or blocking AT2 represent rational strategies to mitigate this common pain syndrome. PERSPECTIVE: This study provides evidence that local inflammation leads to angiotensin II formation, which acts on the AT2 to induce nociceptor axon sprouting in vulvodynia. Preventing inflammation and blocking AT2 therefore present potential pharmacological strategies for reducing vestibular pain.

Cancer early detection program based on awareness and clinical breast examination: Interim results from an urban community in Mumbai, India.

Indian women with breast cancer are usually diagnosed in advanced stages leading to poor survival. Improving breast awareness and increasing access to early diagnosis and adequate treatment has been advocated for breast cancer control. We implemented a program to increase awareness on breast cancer and access to its early detection in an occupational health care scheme in Mumbai, India. Breast awareness brochures were mailed annually between June 2013 and June 2016 to a cohort of 22,500 eligible women aged 30-69 years old receiving universal health care from an occupational health care scheme comprising of primary health centres and a referral secondary care hospital in Mumbai. Women with suspected breast cancers were provided with diagnostic investigations and treatment. Socio-demographic information and tumour characteristics were compared between the breast awareness pre-intervention period (Jan 2005-May 2013) and the breast awareness intervention period after four rounds of mailers (June 2013-June 2016). The proportion of women with early tumours and axillary lymph node negative cancers increased from 74% to 81% and 46% to 53% respectively, between the two periods. While the proportion of patients receiving breast conserving surgery increased from 39% to 51%, the proportion receiving chemotherapy decreased from 84% to 56%. Interim results following efforts to improve breast awareness and access to care in a cohort of women in an occupational health care scheme indicate early detection and more conservative treatment of breast cancers. Creating awareness and improving access to care may result in cancer down-staging.

Angiotensin II receptor type 2 activation is required for cutaneous sensory hyperinnervation and hypersensitivity in a rat hind paw model of inflammatory pain.

UNLABELLED: Many pain syndromes are associated with abnormal proliferation of peripheral sensory fibers. We showed previously that angiotensin II, acting through its type 2 receptor (AT2), stimulates axon outgrowth by cultured dorsal root ganglion neurons. In this study, we assessed whether AT2 mediates nociceptor hyperinnervation in the rodent hind paw model of inflammatory pain. Plantar injection of complete Freund's adjuvant (CFA), but not saline, produced marked thermal and mechanical hypersensitivity through 7 days. This was accompanied by proliferation of dermal and epidermal PGP9.5-immunoreactive (ir) and calcitonin gene-related peptide-immunoreactive (CGRP-ir) axons, and dermal axons immunoreactive for GFRα2 but not tyrosine hydroxylase or neurofilament H. Continuous infusion of the AT2 antagonist PD123319 beginning with CFA injection completely prevented hyperinnervation as well as hypersensitivity over a 7-day period. A single PD123319 injection 7 days after CFA also reversed thermal hypersensitivity and partially reversed mechanical hypersensitivity 3 hours later, without affecting cutaneous innervation. Angiotensin II-synthesizing proteins renin and angiotensinogen were largely absent after saline but abundant in T cells and macrophages in CFA-injected paws with or without PD123319. Thus, emigrant cells at the site of inflammation apparently establish a renin-angiotensin system, and AT2 activation elicits nociceptor sprouting and heightened thermal and mechanical sensitivity. PERSPECTIVE: Short-term AT2 activation is a potent contributor to thermal hypersensitivity, whereas long-term effects (such as hyperinnervation) also contribute to mechanical hypersensitivity. Pharmacologic blockade of AT2 signaling represents a potential therapeutic strategy aimed at biologic mechanisms underlying chronic inflammatory pain.

Hypersensitivity and hyperinnervation of the rat hind paw following carrageenan-induced inflammation.

Studies of human tissue show that many chronic pain syndromes are accompanied by abnormal increases in numbers of peripheral sensory nerve fibers. It is not known if sensory nerve sprouting occurs as a result of inflammation present in these conditions, or other factors such as infection or extensive tissue damage. In the present study, we used a well established model of inflammation to examine cutaneous innervation density in relation to mechanical and thermal hypersensitivity. Adult female rats were ovariectomized to eliminate fluctuations in female reproductive hormones and one week later, a hind paw was injected with carrageenan or saline vehicle. Behavioral testing showed that saline vehicle injection did not alter thermal or mechanical thresholds compared to pre-injection baselines. Carrageenan injections resulted in markedly reduced paw withdrawal thresholds at 24 and 72 h after injection; this was accompanied by increased mechanical sensitivity of the contralateral paw at 72 h. Analysis of innervation density using PGP9.5 as a pan-neuronal marker at 72 h showed that inflammation resulted in a 2-fold increase in cutaneous innervation density. We conclude that inflammation alone is sufficient to induce sprouting of sensory cutaneous axon endings leading local tissue hyperinnervation, which may contribute to hypersensitivity that occurs in painful inflammatory conditions.

Estrogen elicits dorsal root ganglion axon sprouting via a renin-angiotensin system.

Many painful conditions occur more frequently in women, and estrogen is a predisposing factor. Estrogen may contribute to some pain syndromes by enhancing axon outgrowth by sensory dorsal root ganglion (DRG) neurons. The objective of the present study was to define mechanisms by which estrogen elicits axon sprouting. The estrogen receptor-alpha agonist propyl pyrazole triol induced neurite outgrowth from cultured neonatal DRG neurons, whereas the estrogen receptor-beta agonist diarylpropionitrile was ineffective. 17beta-Estradiol (E2) elicited sprouting from peripherin-positive unmyelinated neurons, but not larger NF200-positive myelinated neurons. Microarray analysis showed that E2 up-regulates angiotensin II (ANGII) receptor type 2 (AT2) mRNA in vitro, and studies in adult rats confirmed increased DRG mRNA and protein in vivo. AT2 plays a central role in E2-induced axon sprouting because AT2 blockade by PD123,319 eliminated estrogen-mediated sprouting in vitro. We assessed whether AT2 may be responding to locally synthesized ANGII. DRG from adult rats expressed mRNA for renin, angiotensinogen, and angiotensin converting enzyme (ACE), and protein products were present and occasionally colocalized within neurons and other DRG cells. We determined if locally synthesized ANGII plays a role in estrogen-mediated sprouting by blocking its formation using the ACE inhibitor enalapril. ACE inhibition prevented estrogen-induced neuritogenesis. These findings support the hypothesis that estrogen promotes DRG nociceptor axon sprouting by up-regulating the AT2 receptor, and that locally synthesized ANGII can induce axon formation. Therefore, estrogen may contribute to some pain syndromes by enhancing the pro-neuritogenic effects of AT2 activation by ANGII.

Pathophysiological manifestations in mice exposed to anthrax lethal toxin.

Pathophysiological changes associated with anthrax lethal toxin included loss of plasma proteins, decreased platelet count, slower clotting times, fibrin deposits in tissue sections, and gross and histopathological evidence of hemorrhage. These findings suggest that blood vessel leakage and hemorrhage lead to disseminating intravascular coagulation and/or circulatory shock as an underlying pathophysiological mechanism.

Quantifying immunohistochemical staining of phospho-eIF2alpha, heme oxygenase-2 and NADPH cytochrome P450 reductase in oligodendrocytes during experimental autoimmune encephalomyelitis.

As a consequence of inflammation associated with multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), stress responses are induced in many cells within the CNS, however, those that occur within the primary pathological target, the oligodendrocyte, are not fully established. Recently, we found that phosphorylated eukaryotic initiation factor-2alpha (eIF2alpha), an inhibitor of protein translation associated with the stress response, is expressed in a greater number of oligodendrocytes in EAE animals compared to controls. However, since numerous oligodendrocytes in control animals also expressed phospho-eIF2alpha, a method was developed to detect expression levels within oligodendrocytes that did not rely on the number of oligodendrocytes that were stained. This method utilized a high dilution of the primary antibody so that the staining density was kept below a maximum plateau which could eliminate expression differences. Furthermore, the staining density within oligodendrocytes, as determined by image analysis, was corrected by the background density or that within neurons. In either case, the density of staining was greater in oligodendrocytes from EAE animals versus controls. The expression of heme oxygenase-2 and NADPH cytochrome P450 reductase also were examined, but unlike phospho-eIF2alpha, neither was increased in oligodendrocytes from EAE animals compared to controls. In summary, a protocol involving a high dilution of primary antibody and image analysis revealed that the expression of phospho-eIF2alpha within oligodendrocytes was increased in EAE animals compared to control animals.

Immunohistochemical localization of phosphorylated protein kinase R and phosphorylated eukaryotic initiation factor-2 alpha in the central nervous system of SJL mice with experimental allergic encephalomyelitis.

Inflammatory cells enter the CNS and target myelin in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), a model of MS, and inflammation is thought to induce stress responses in the CNS. Protein kinase R (PKR) and eukaryotic initiation factor-2 alpha (eIF2 alpha) undergo phosphorylation in response to stress, and the phosphorylated forms of these proteins play a key role in regulating protein synthesis. The objective of this study was to investigate the expression profile of phospho-PKR and phospho-eIF2 alpha during the course of EAE in order to advance the understanding of the stress response in this disease. In control animals (no encephalitogen with no emulsion; no encephalitogen with emulsion) and in preclinical EAE animals, phospho-PKR immunoreactivity was present in oligodendrocytes and some neurons, whereas, in EAE animals with active disease there was widespread labeling of inflammatory cells, and these cells were present during the recovery period of EAE, albeit to a lesser extent. Double-labeling studies revealed that T cells and a few macrophages were phospho-PKR(+). Phospho-eIF2 alpha immunoreactivity was detected in some oligodendrocytes in hindbrain sections of control animals. In EAE animals with active disease, the number of labeled oligodendrocytes increased, and inflammatory T cells also were labeled. Insofar as phospho-PKR activates nuclear factor-kappa B, it may facilitate cytokines expression by T cells. Alternatively, phospho-PKR and phospho-eIF2 alpha may promote apoptosis as a way to regulate T-cell number in the CNS. The expression of phospho-eIF2 alpha in oligodendrocytes during EAE likely is involved with inhibition of protein translation, which is a protective mechanism used to promote cell survival in response to inflammation.

The role of iron in the pathogenesis of experimental allergic encephalomyelitis and multiple sclerosis.

Multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE), are autoimmune disorders resulting in demyelination in the central nervous system (CNS). Pathologically, the blood-brain barrier becomes damaged, macrophages and T cells enter into the CNS, oligodendrocytes and myelin are destroyed, astrocytes and microglia undergo gliosis, and axons become transected. Data from several biochemical and pharmacological studies indicate that free radicals participate in the pathogenesis of EAE, and iron has been implicated as the catalyst leading to their formation. The primary focus of this article is the examination of the role of iron in the pathogenesis of MS and EAE. Particular attention will be paid to the role and distribution of iron and proteins involved with iron metabolism (e.g., transferrin, ferritin, heme oxygenase-1, etc.) in normal and disease states of myelin. Furthermore, therapeutic interventions aimed at iron, iron-binding proteins, and substrates or products of iron-catalyzed reactions leading to free radical production will be discussed.