Anna Maria Di Giulio

Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma

Erythropoietin (EPO) functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production. In the brain, for example, EPO and its receptor are locally produced, are modulated by metabolic stressors, and provide neuroprotective and antiinflammatory functions. We have previously shown that recombinant human EPO (rhEPO) administered within the systemic circulation enters the brain and is neuroprotective. At present, it is unknown whether rhEPO can also improve recovery after traumatic injury of the spinal cord. To evaluate whether rhEPO improves functional outcome if administered after cord injury, two rodent models were evaluated. First, a moderate compression of 0.6 N was produced by application of an aneursym clip at level T3 for 1 min. RhEPO (1,000 units per kg of body weight i.p.) administered immediately after release of compression was associated with partial recovery of motor function within 12 h after injury, which was nearly complete by 28 days. In contrast, saline-treated animals exhibited only poor recovery. In the second model used, rhEPO administration (5,000 units per kg of body weight i.p. given once 1 h after injury) also produced a superior recovery of function compared with saline-treated controls after a contusion of 1 N at level T9. In this model of more severe spinal cord injury, secondary inflammation was also markedly attenuated by rhEPO administration and associated with reduced cavitation within the cord. These observations suggest that rhEPO provides early recovery of function, especially after spinal cord compression, as well as longer-latency neuroprotective, antiinflammatory and antiapoptotic functions.

Significance of dopamine metabolites in the evaluation of drugs acting on dopaminergic neurones

The effect of various drugs was studied on 3-methoxytyramine (3-MT) concentrations in rat striatum. The drugs were chosen for their ability to interfere with the dopaminergic system at different levels. Dopamine (DA) acidic metabolites, i.e. homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), were also measured. Changes of 3-MT, unlike those of DOPAC and HVA, seem to reflect the functional activity of dopaminergic neurons. In fact drugs believed to increase or decrease DA content in the synaptic cleft produce predictable changes of striatal 3-MT. Thus cocaine, nomifensine and d-amphetamine increase 3-MT concentrations while gamma-butyrolactone, alpha-methyltyrosine and apomorphine decrease it.

Enkephalin in bovine adrenal gland: multiple molecular forms of [Met5]-enkephalin immunoreactive peptides.

Abstract The existence and heterogeneity of enkephalin-like substances in bovine adrenal glands has been demonstrated using enkephalin radioimmunoassays. Both [met5]- and [leu5]-enkephalins were found to be highly enriched in the medulla i.e. 6.65 pmol [met5]-enkephalin/mg protein but only a very small amount, 0.041 pmol [met5]-enkephalin/mg protein, was detected in the cortex. In addition to the pentapeptide, [met5]-enkephalin, a large proportion of the total [met5]-enkephalin immunoreactive substance was found by gel filtration chromatography to be present in two high molecular weight forms. Determination of the true concentration of the high molecular weight forms, however, awaits purification and use of a proper standard. The existence of the different molecular forms of [met5]-enkephalin immunoreactive substances was substantiated by immunoabsorption experiment and gel filtration chromatography with 6 M guanidine. The two high molecular weight immunoreactive substances were not assayable by the radioreceptor binding assay; however, when trypsinized, they released a product which was capable of binding to the opiate receptor and was indistinguishable from the pentapeptide. [met5]-enkephalin, with respect to its size and thin layer Chromatographic characteristic. Whether the high molecular weight [met5]-enkephalin immunoreactive substances can function as precursors of [met5]-enkephalin in adrenal glands remains to be determined.

Systemic administration of insulin‐like growth factor decreases motor neuron cell death and promotes muscle reinnervation

Neonatal sciatic nerve axotomy causes motoneuron death and muscle denervation atrophy. The aim of the present study was to determine whether insulin‐like growth factor‐I (IGF‐I) administration promotes muscle reinnervation and counteracts motor neuron loss after such an injury. Six weeks after sciatic nerve axotomy performed in 2‐day‐old pups, the number of motor neurons, as assessed by retrograde transport of horseradish peroxidase injected into the extensor digitorum longus (EDL) muscle, was reduced from 52 ± 3 to 26 ± 3. Subsequent administration of IGF‐I at the doses of 0.02 mg/kg or 1 mg/kg increased the number of motor neurons to 35 ± 2 and 37 ± 5, respectively. The effect on motoneuron survival was accompanied by improved muscle fibre morphometry and restoration of indirect EDL muscle isometric twitch tension, which was about 80% of control values for both doses of IGF‐I compared with 60% observed with saline treatment. Reinnervated EDL muscle from saline‐treated rats cannot hold tetanic tension, which is, however, achieved after IGF‐I treatment at either dose. Thus, both high and low doses of IGF‐I counteracted motoneuron death and improved muscle reinnervation following neonatal sciatic nerve axotomy. IGF‐I at 5 μg/kg failed to increase muscle reinnervation. J. Neurosci. Res. 54:840–847, 1998.

Reparixin, an Inhibitor of CXCR2 Function, Attenuates Inflammatory Responses and Promotes Recovery of Function after Traumatic Lesion to the Spinal Cord

It has been shown that the blockade of CXCR1 and CXCR2 receptors prevents ischemia/reperfusion damage in several types of vascular beds. Reparixin is a recently described inhibitor of human CXCR1/R2 and rat CXCR2 receptor activation. We applied reparixin in rats following traumatic spinal cord injury and determined therapeutic temporal and dosages windows. Treatment with reparixin significantly counteracts secondary degeneration by reducing oligodendrocyte apoptosis, migration to the injury site of neutrophils and ED-1-positive cells. The observed preservation of the white matter might also be secondary to the enhanced proliferation of NG2-positive cells. The expression of macrophage-inflammatory protein-2, tumor necrosis factor-α, interleukin (IL)-6, and IL-1β was also counteracted, and the proliferation of glial fibrillary acidic protein-positive cells was markedly reduced. These effects resulted in a smaller post-traumatic cavity and in a significantly improved recovery of hind limb function. The best beneficial outcome of reparixin treatment required 7-day administration either by i.p. route (15 mg/kg) or subcutaneous infusion via osmotic pumps (10 mg/kg), reaching a steady blood level of 8 μg/ml. Methylprednisolone was used as a reference drug; such treatment reduced cytokine production but failed to affect the rate of hind limb recovery.

Embryonic stem cells promote motor recovery and affect inflammatory cell infiltration in spinal cord injured mice.

The purpose of this study was to determine the fate and the effects of undifferentiated embryonic stem cells (ESCs) in mice after contusive lesion of the spinal cord (SCI). Reproducible traumatic lesion to the cord was performed at T8 level by means of the Infinite Horizon Device, and was followed by intravenous injection of one million of undifferentiated ESCs through the tail vein within 2 h from the lesion. The ESCs-treated animals showed a significant improvement of the recovery of motor function 28 days after lesion, with an average score of 4.61+/-0.13 points of the Basso Mouse Scale (n=14), when compared to the average score of vehicle treated mice, 3.58+/-0.23 (n=10). The number of identified ESCs found at the lesion site was 0.6% of the injected cells at 1 week after transplantation, and further reduced to 0.04% at 1 month. It is, thus, apparent that the promoted hind-limb recovery cannot be correlated to a substitution of the lost tissue performed by the exogenous ESC. The extensive evaluation of production of several neuroprotective and inflammatory cytokines did not reveal any effect by ESC-treatment, but unexpectedly the number of invading macrophages and neutrophils was greatly reduced. This may explain the improved preservation of lesion site ventral myelin, at both 1 week (29+/-11%) and 1 month (106+/-14%) after injury. No teratoma formation was observed, although an inappropriate colonization of the sacral cord by differentiated nestin- and beta-tubulin III-positive ESCs was detected.

Effects of low doses of glycosaminoglycans and insulin-like growth factor-I on motor neuron disease in wobbler mouse

In this study we examined the effects of insulin-like growth factor-I (IGF-I) and of glycosaminoglycans (GAGs) on the progressive motor neuron disease in wobbler mice. After clinical diagnosis at age 3 weeks, mice received daily subcutaneous injections of IGF-I, or GAGs, or saline for 3 weeks. The histometric analysis revealed that biceps muscle fiber diameter was reduced in wobbler mice and that treatments with GAGs and IGF-I prevented such a drop. The number of atrophic small fibers was markedly reduced and that of the larger ones augmented. No effects on body growth and biceps muscle weight were observed. The combined AChE-silver staining revealed that both treatments promoted intramuscular axonal sprouting. The typical decline of grip strength in wobbler mice was also prevented. This study suggests that GAGs and IGF-I administrations can retard the onset of motor deficit, and reduce muscle atrophy in wobbler mice.

Adult neural precursors isolated from post mortem brain yield mostly neurons: An erythropoietin-dependent process

This study was aimed at the isolation of neural precursor cells (NPCs) capable of resisting to a prolonged ischemic insult as this may occur at the site of traumatic and ischemic CNS injuries. Adult mice were anesthetized and then killed by cervical dislocation. The cadavers were maintained at room temperature or at 4°C for different time periods. Post mortem neural precursors (PM-NPCs) were isolated, grown in vitro and their differentiation capability was investigated by evaluating the expression of different neuronal markers. PM-NPCs differentiate mostly in neurons, show activation of hypoxia-inducible factor-1 and MAPK, and express both erythropoietin (EPO) and its receptor (EPO-R). The exposure of PM-NPCs to neutralizing antibodies to EPO or EPO-R dramatically reduced the extent of neuronal differentiation to about 11% of total PM-NPCs. The functionality of mTOR and MAPK is also required for the expression of the neuronal phenotype by PM-NPCs. These results suggest that PM-NPCs can be isolated from animal cadaver even several hours after death and their self-renewable capability is comparable to normal neural precursors. Differently, their ability to achieve a neural phenotype is superior to that of NPCs, and this is mediated by the activation of hypoxia-induced factor 1 and EPO signaling. PM-NPCs may represent good candidates for transplantation studies in animal models of neurodegenerative diseases.

The Ribonucleic Complex HuR-MALAT1 Represses CD133 Expression and Suppresses Epithelial–Mesenchymal Transition in Breast Cancer

Epithelial-to-mesenchymal transition (EMT) is a core process underlying cell movement during embryonic development and morphogenesis. Cancer cells hijack this developmental program to execute a multi-step cascade, leading to tumorigenesis and metastasis. CD133 (PROM1), a marker of cancer stem cells, has been shown to facilitate EMT in various cancers, but the regulatory networks controlling CD133 gene expression and function in cancer remain incompletely delineated. In this study, we show that a ribonucleoprotein complex including the long noncoding RNA MALAT1 and the RNA-binding protein HuR (ELAVL1) binds the CD133 promoter region to regulate its expression. In luminal nonmetastatic MCF-7 breast cancer cells, HuR silencing was sufficient to upregulate N-cadherin (CDH2) and CD133 along with a migratory and mesenchymal-like phenotype. Furthermore, we found that in the basal-like metastatic cell line MDA-MB-231 and primary triple-negative breast cancer tumor cells, the repressor complex was absent from the CD133-regulatory region, but was present in the MCF-7 and primary ER+ tumor cells. The absence of the complex from basal-like cells was attributed to diminished expression of MALAT1, which, when overexpressed, dampened CD133 levels. In conclusion, our findings suggest that the failure of a repressive complex to form or stabilize in breast cancer promotes CD133 upregulation and an EMT-like program, providing new mechanistic insights underlying the control of prometastatic processes. Cancer Res; 76(9); 2626-36. ©2016 AACR.

Third trimester amniotic fluid cells with the capacity to develop neural phenotypes and with heterogeneity among sub-populations

PURPOSE Our aim was the search for new sources of cells potentially useful for central nervous system regenerative medicine. Extra-embryonic tissues are promising sources of pluripotent stem cells. Among these, human second-trimester amniotic fluid (AF) contains cell populations exhibiting self-renewal capacity, multipotency and the expression of embryonic cell markers. METHODS Here we report the properties of the easily available third-trimester AF cells (AFCs). Different cell types from 6 of 9 AF samples were separated, expanded, and characterized by assessing their morphological, proliferative, and differentiative properties. RESULTS All isolated cultures presented CD105, CD90 and CD73 mesenchymal markers, whereas they differed among themselves in CD117, CD146, CD31, NG2 and CD133 expression. Their doubling time and telomere length were conserved throughout many passages. Importantly, immunofluorescence and Real-time PCR showed that, during their proliferative state and differentiation, several cultures expressed neuronal and glial markers such as nestin, GFAP, β-tubulin III and neurofilament H indicating their potential attitude towards a neural fate. Indeed, these cells showed a rather poor capacity to differentiate in adipogenic and osteogenic lineages. CONCLUSIONS In this work we report that cells with neural differentiation capability can be isolated from third-trimester AF, such properties could be useful for neuro-regenerative purposes.