Cynthia J.M. Kane

Activated Macrophage/Microglial Cells Can Promote the Regeneration of Sensory Axons into the Injured Spinal Cord

A prominent role for phagocytic cells in the regenerative response to CNS or PNS injury has been suggested by numerous studies. In the present work we tested whether increasing the presence of phagocytic cells at a spinal cord injury site could enhance the regeneration of sensory axons from cut dorsal roots. Nitrocellulose membranes treated with TGF-beta or coated with microglial cells were cotransplanted with fetal spinal cord tissue into an injured adult rat spinal cord. Cut dorsal roots were apposed to both sides of the nitrocellulose. Four weeks later, animals were sacrificed and spinal cord tissue sections were processed for immunocytochemical detection of calcitonin gene-related peptide (CGRP-ir) to identify regenerated sensory axons. Adjacent sections were processed with the antibody ED-1 or the lectin GSA-B4 for detection of macrophage/microglial cells in association with the regrowing axons. Qualitative and quantitative data indicate a correlation between the pattern and extent of axonal regeneration and the presence of phagocytic cells along the nitrocellulose implant. Axonal regeneration could be experimentally limited by implanting a nitrocellulose strip treated with macrophage inhibitory factor. These results indicate that increasing the presence of activated macrophage/microglial cells at a spinal cord injury site can provide an environment beneficial to the promotion of regeneration of sensory axons, possibly by the release of cytokines and interaction with other nonneuronal cells in the immediate vicinity.

Platelet-rich plasma gel promotes differentiation and regeneration during equine wound healing

Nonhealing wounds of the lower equine limb represent a challenging model. The platelet is a natural source of a myriad of growth factors and cytokines that promote wound healing. This study evaluates the potential of platelet derived factors to enhance wound healing in the lower equine limb. Platelets were isolated from horse blood and activated with thrombin, a process known to induce growth factor release. This produced a platelet gel composed of platelet-rich plasma (PRP). To test this all-natural wound healant, 2.5-cm(2) full thickness cutaneous wounds were created below the knee and hock of a thoroughbred horse. Wounds were treated with PRP gel or left untreated. Sequential wound biopsies collected at Days 7, 36, and 79 postwounding permitted comparison of the temporal expression of differentiation markers and wound repair. To test the hypothesis that wounds treated with PRP gel exhibit more rapid epithelial differentiation and enhanced organization of dermal collagen compared to controls, tissues were stained for cytokeratin 10, a suprabasal differentiation marker, and the reestablishment of collagen was evaluated by trichrome staining. PRP gel-treated wounds at Day 7 expressed intense cytokeratin 10 staining near the wound junction in suprabasal epidermal layers, while staining in control tissues was less intense and restricted to apical epidermal layers distal to the wound junction. By Day 79, the staining was equal in both groups. However, PRP gel-treated wounds at Day 79 contained abundant, dense collagen bundles oriented parallel to each other and to the overlying epithelium, whereas control tissues contained fewer collagen fibers that were oriented randomly. Thus, treatment of wounds with PRP gel induced accelerated epithelial differentiation and produced tissue with organized, interlocking collagen bundles. This study reveals that this novel all-natural wound healant induced wound repair in injuries previously deemed untreatable.

Expression of fibrogenic cytokines in rat small intestine after fractionated irradiation

The molecular and cellular mechanisms that regulate the radiation-induced fibrotic response in the intestine are not known. In addition to increased amounts of connective tissue, inflammatory cell aggregates are often found, especially in conjunction with acute or chronic mucosal ulcerations. These inflammatory cells are a major source of cytokines that influence connective tissue metabolism. Hence, a possible link may exist between the cellular inflammatory response and fibrosis. This preclinical study examined the influence of fractionated irradiation on the expression of three inflammatory/fibrogenic cytokines in rat small intestine. A rat intestinal transposition model was used for localized fractionated irradiation of a 3-4-cm segment of small bowel. Fifty-nine male Sprague-Dawley rats were irradiated or sham irradiated with 9 daily fractions of 5.2 Gy. Expression of Interleukin 1 alpha (IL-1 alpha), Transforming growth factor beta 1 (TGF-beta 1), and Platelet derived growth factor-AA (PDGF-AA) was assessed by immunohistochemistry. Irradiated and unirradiated intestine was examined 24 h, 14 days, and 26 weeks after completion of irradiation. Unirradiated intestine exhibited immunohistochemical expression of IL-1 alpha, TGF-beta 1 and PDGF-AA that conformed to known staining patterns in normal tissue. Irradiated intestine showed increased expression of all three cytokines at all assessment times. The increased cytokine expression correlated with fibrosis and inflammatory cell infiltrates in irradiated intestine. This was particularly evident in areas with mucosal ulcerations. Fractionated irradiation of small intestine elicits increased expression of IL-1 alpha, TGF-beta 1, and PDGF-AA in areas of acute and chronic radiation injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of neurons and microglia against ethanol in a mouse model of fetal alcohol spectrum disorders by peroxisome proliferator-activated receptor-γ agonists.

Fetal alcohol spectrum disorders (FASD) result from ethanol exposure to the developing fetus and are the most common cause of mental retardation in the United States. These disorders are characterized by a variety of neurodevelopmental and neurodegenerative anomalies which result in significant lifetime disabilities. Thus, novel therapies are required to limit the devastating consequences of FASD. Neuropathology associated with FASD can occur throughout the central nervous system (CNS), but is particularly well characterized in the developing cerebellum. Rodent models of FASD have previously demonstrated that both Purkinje cells and granule cells, which are the two major types of neurons in the cerebellum, are highly susceptible to the toxic effects of ethanol. The current studies demonstrate that ethanol decreases the viability of cultured cerebellar granule cells and microglial cells. Interestingly, microglia have dual functionality in the CNS. They provide trophic and protective support to neurons. However, they may also become pathologically activated and produce inflammatory molecules toxic to parenchymal cells including neurons. The findings in this study demonstrate that the peroxisome proliferator-activated receptor-γ agonists 15-deoxy-Δ12,15 prostaglandin J2 and pioglitazone protect cultured granule cells and microglia from the toxic effects of ethanol. Furthermore, investigations using a newly developed mouse model of FASD and stereological cell counting methods in the cerebellum elucidate that ethanol administration to neonates is toxic to both Purkinje cell neurons as well as microglia, and that in vivo administration of PPAR-γ agonists protects these cells. In composite, these studies suggest that PPAR-γ agonists may be effective in limiting ethanol-induced toxicity to the developing CNS.

Pioglitazone Blocks Ethanol Induction of Microglial Activation and Immune Responses in the Hippocampus, Cerebellum, and Cerebral Cortex in a Mouse Model of Fetal Alcohol Spectrum Disorders

BACKGROUND Fetal alcohol spectrum disorders (FASD) result from fetal exposure to alcohol and are the leading cause of mental retardation in the United States. There is currently no effective treatment that targets the causes of these disorders. Thus, novel therapies are critically needed to limit the neurodevelopmental and neurodegenerative pathologies associated with FASD. METHODS A neonatal mouse FASD model was used to examine the role of the neuroimmune system in ethanol (EtOH)-induced neuropathology. Neonatal C57BL/6 mice were treated with EtOH, with or without pioglitazone, on postnatal days 4 through 9, and tissue was harvested 1 day post treatment. Pioglitazone is a peroxisome proliferator-activated receptor (PPAR)-γ agonist that exhibits anti-inflammatory activity and is neuroprotective. We compared the effects of EtOH with or without pioglitazone on cytokine and chemokine expression and microglial morphology in the hippocampus, cerebellum, and cerebral cortex. RESULTS In EtOH-treated animals compared with controls, cytokines interleukin-1β and tumor necrosis factor-α mRNA levels were increased significantly in the hippocampus, cerebellum, and cerebral cortex. Chemokine CCL2 mRNA was increased significantly in the hippocampus and cerebellum. Pioglitazone effectively blocked the EtOH-induced increase in the cytokines and chemokine in all tissues to the level expressed in handled-only and vehicle-treated control animals. EtOH also produced a change in microglial morphology in all brain regions that was indicative of microglial activation, and pioglitazone blocked this EtOH-induced morphological change. CONCLUSIONS These studies indicate that EtOH activates microglia to a pro-inflammatory stage and also increases the expression of neuroinflammatory cytokines and chemokines in diverse regions of the developing brain. Further, the anti-inflammatory and neuroprotective PPAR-γ agonist pioglitazone blocked these effects. It is proposed that microglial activation and inflammatory molecules expressed as a result of EtOH treatment during brain development contribute to the sequelae associated with FASD. Thus, pioglitazone and anti-inflammatory pharmaceuticals more broadly have potential as novel therapeutics for FASD.

Ethanol-induced alterations of neurotrophin receptor expression on Purkinje cells in the neonatal rat cerebellum

Ethanol causes loss of Purkinje cells in the cerebellum during the early stages of differentiation and maturation by a presently unknown mechanism. Neuronal vulnerability in the cerebellum parallels the prominent temporal and anatomical gradients of development (i.e. early to late interlobular and posterior to anterior, respectively). Development of Purkinje cells is known to require binding of the neurotrophins, including brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), to the tyrosine-kinase (Trk) receptors TrkB and TrkC, respectively. In addition, Purkinje cells are reported to experience a critical switch between BDNF dependence and NT3 dependence during the period of highest ethanol sensitivity between postnatal days (PN) 4-6. To test the hypothesis that ethanol alters neurotrophin signaling leading to Purkinje neuronal death, the immunohistochemical expression of TrkB and TrkC receptors on Purkinje cells of rat pups following a moderate dose of ethanol was determined at various times surrounding the period of postnatal ethanol vulnerability. Ethanol selectively decreased Purkinje cell expression of TrkB and TrkC receptors following exposures within the vulnerable period (PN4-6). These results suggest that ethanol may induce loss of Purkinje cells by alteration of neurotrophic regulation at this critical stage.

Methyl gallate, methyl-3,4,5-trihydroxybenzoate, is a potent and highly specific inhibitor of herpes simplex virusin vitro. II. Antiviral activity of methyl gallate and its derivatives

Methyl gallate (MG), methyl-3,4,5-trihydroxybenzoate, was highly active against herpes viruses as determined by plaque reduction assay. Herper simplex virus type 2, MS strain, was sensitive to MG at a mean 50% inhibitory concentration (IC50) of 0.224 μg/ml in monkey kidney cells. MG was specific for herpes viruses with the relative sensitivity HSV-2>HSV-1>CMV. Two RNA viruses tested were significantly less sensitive to MG. The structural components of MG which modulate the anti-herpetic activity were identified by analysis of chemical analogues. Our structural analyses indicated that three hydroxyl groups were required but were not sufficient for the anti-herpetic action of MG. The presence and chain length of the alkyl ester were also important to the anti-herpetic activity of MG. Methyl gallate may interact with virus proteins and alter the adsorption and penetration of the virion.

Therapeutic potential of natural compounds that regulate the activity of protein kinase C.

Protein kinase C (PKC) is a family of serine/threonine kinases that regulates a variety of cell functions including proliferation, gene expression, cell cycle, differentiation, cytoskeletal organization, cell migration, and apoptosis. The PKC signal transduction cascade coordinates complex physiological events including normal tissue function and repair. Disruption of the cellular environment through genetic mutation, disease, injury, or exposure to pro-oxidants, alcohol, or other insults can induce pathological PKC activation. Aberrant PKC activation can lead to diseases of cellular dysregulation such as cancer and diabetes. Can aberrant activation of PKC be reversed? Even 25 years after the identification of PKC, therapeutic regulation of PKC activity remains an emerging field. Because the function of each isoform remains to be elucidated, isoform specific control of gene expression is a current challenge. Natural compounds are important regulators of PKC activity, with both preventive and therapeutic efficacy. Antioxidants including vitamin A (retinoids), vitamin C (ascorbic acid) and vitamin E (tocopherols) show promise for reversal of PKC activation. beta-carotene and retinoids function as anticarcinogenic agents and antagonize the biological effects of pro-oxidants on PKC. Vitamin E reverses the deleterious effects of hyperglycemia and diabetes by down-regulating PKC activity. Antioxidants in red wine provide cardioprotective effects. However, alcohol consumption also induces oxidative stress and disrupts PKC and retinoid function in the fetus and the adult. This review examines modulation of PKC activity by natural compounds and pharmacologic analogues which can be used effectively to prevent or treat common diseases associated with aberrant activation of PKC.

Proliferation of astroglia from the adult human cerebrum is inhibited by ethanol in vitro

Chronic alcoholism is associated with atrophy of the adult brain, while fetal exposure to ethanol can cause microencephaly. Since astroglial pathology is a common feature of ethanol exposure in both humans and animal models, the direct influence of ethanol on proliferation of human astroglia from the gray and white matter of adult temporal lobe was determined and compared. Astroglial cultures were exposed to constant concentrations of ethanol at realistic social and clinical levels (0.1, 0.2 or 0.5%; w/v) for 1 to 5 days. Proliferation was quantified by bromodeoxyuridine labeling and enumeration of replicating cells. Ethanol exposure significantly inhibited proliferation of both gray and white matter astroglia in a dose and duration dependent manner. Gray matter was slightly more sensitive than white matter to inhibition by low to moderate concentrations of ethanol; in contrast, white matter was more sensitive to high ethanol concentrations. Maximum inhibition was 20% in gray matter and 25% in white matter. Human astroglial proliferation was directly inhibited in the absence of neurons, microglia, neuronal degeneration or systemic factors that have confounded in vivo studies. Restricted astroglial proliferation may underlie aspects of the astroglial pathology associated with ethanol exposure.

Genomic Heterogeneity of DNA Repair

The introduction and repair of DNA lesions are generally heterogeneous with respect to different genomic domains. In particular, the repair of helix-distorting damage, such as the cyclobutane pyrimidine dimers (CPD) induced by ultraviolet light occurs selectively in expressed genes. This is due in large part to the preferential repair of transcribed DNA strands, which is then reflected in a bias toward mutagenesis from persisting lesions in nontranscribed strands. Consequently, determination of overall genomic repair efficiencies may not be a good indicator of cellular sensitivity to agents that damage DNA. Although some studies suggest an age-related accumulation of altered nucleotides in DNA, we do not know the intragenomic distribution of those changes and whether they are relevant to the physiological aspects of aging. Subtle changes in the pattern of preferential repair during maturation could have profound effects on cell and tissue function. DNA repair has been analyzed in differentiating cell systems as possible models for aging. We have observed attenuated overall repair of CPD in differentiated rat myoblasts or PC12 neuron-like cells. In both model systems, several expressed genes have been shown to be repaired relatively efficiently but without strand specificity. In another model system of human HT1080 fibroblasts differentiating in the presence of dexamethasone, we demonstrated enhanced repair in the gene for plasminogen activator inhibitor I whose transcription is induced and, correspondingly, a reduced repair rate in the urokinase plasminogen activator gene whose transcription is suppressed. We conclude that any attempted correlation of the phenomena of aging with DNA repair should focus on the relevant genes in the tissue of interest.