Anuradha Chakrabarty

The Role of Iron in the Pathogenesis of Experimental Allergic Encephalomyelitis and Multiple Sclerosis

Abstract: 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.

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

ABSTRACT 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.

Immunohistochemical localization of phosphorylated protein kinase R and phosphorylated eukaryotic initiation factor-2α 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‐2α (eIF2α) 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‐eIF2α 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‐eIF2α 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‐κB, it may facilitate cytokines expression by T cells. Alternatively, phospho‐PKR and phospho‐eIF2α may promote apoptosis as a way to regulate T‐cell number in the CNS. The expression of phospho‐eIF2α 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.

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.

Quantifying immunohistochemical staining of phospho-eIF2α, 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.

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 Freunds 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 Freunds 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.