Rosario Giuffrida

Differential Expression of Estrogen Receptors Alpha and Beta in the Spinal Cord during Postnatal Development: Localization in Glial Cells

Estrogens are recognized as neuroprotective and neurotrophic agents in the central nervous system. They are involved in neuronal differentiation and survival and promote neural development. Estrogen receptors alpha (ER-α) and beta (ER-β) are predominantly expressed in neurons, whereas their presence in glial cells in vivo is more controversial. Changes in their expression during development have been described in different brain areas, but little is known about their presence in the spinal cord. We have carried out an immunohistochemical study in an attempt to analyze the expression of both ERs in astrocytes and oligodendrocytes of the rat spinal cord and their modifications during postnatal development. RT-PCR analysis of whole spinal cord extracts from 4-, 12-, and 25-day-old and adult rats indicated changes in the expression of both receptors during maturation. Immunohistochemistry of slices of the lumbar tract revealed that in an area of the ventral spinal cord that does not contain neuronal cell bodies, but mainly fibers and glial cells, both ER-α and ER-β can be detected. Immunostaining is clearly nuclear, and, in the case of ER-α, both markedly positive and weakly labeled cells can be identified. ER-α is expressed during early development to progressively decline in the adult stage. In contrast, the ER-β signal is low and peaks at postnatal day 25, whereas it is almost undetectable at other ages. Colocalization studies revealed that, at postnatal day 25, ER-α and ER-β are expressed in astrocytes (identified by the specific marker glial fibrillar acidic protein) and oligodendrocytes (labeled by antimyelin 2′,3′-cyclic nucleotide 3′-phosphodiesterase). The present results confirm the expression of ER-α and ER-β in glial cells in vivo and suggest that, also in the spinal cord, glial cells may contribute to the effects of estrogen during development.

Mesenchymal stem cells from adipose tissue which have been differentiated into chondrocytes in three-dimensional culture express lubricin

The present study focused on the isolation, cultivation and characterization of human mesenchymal stem cells (MSCs) from adipose tissue and on their differentiation into chondrocytes through the NH ChondroDiff medium. The main aim was to investigate some markers of biomechanical quality of cartilage, such as lubricin, and collagen type I and II. Little is known, in fact, about the ability of chondrocytes from human MSCs of adipose tissue to generate lubricin in three-dimensional (3D) culture. Lubricin, a 227.5-kDa mucinous glycoprotein, is known to play an important role in articular joint physiology, and the loss of accumulation of lubricin is thought to play a role in the pathology of osteoarthritis. Adipose tissue is an alternative source for the isolation of multipotent MSCs, which allows them to be obtained by a less invasive method and in larger quantities than from other sources. These cells can be isolated from cosmetic liposuctions in large numbers and easily grown under standard tissue culture conditions. 3D chondrocytes were assessed by histology (hematoxylin and eosin) and histochemistry (Alcian blue and Safranin-O/fast green staining). Collagen type I, II and lubricin expression was determined through immunohistochemistry and Western blot. The results showed that, compared with control cartilage and monolayer chondrocytes showing just collagen type I, chondrocytes from MSCs (CD44-, CD90- and CD105- positive; CD45-, CD14- and CD34-negative) of adipose tissue grown in nodules were able to express lubricin, and collagen type I and II, indicative of hyaline cartilage formation. Based on the function of lubricin in the joint cavity and disease and as a potential therapeutic agent, our results suggest that MSCs from adipose tissue are a promising cell source for tissue engineering of cartilage. Our results suggest that chondrocyte nodules producing lubricin could be a novel biotherapeutic approach for the treatment of cartilage abnormalities.

Metabotropic glutamate receptor expression in cultured rat astrocytes and human gliomas

In order to confirm the existence of metabotropic glutamate receptors in astroglial cultures and to provide information on different receptor subtypes, the expression of different mOIuRs was analysed in cultures highly enriched in rat astroglial cells. mRNA levels for mGluR1, 2, 3, 4, 7 were undetectable by Northern blot analysis in primary type-1 astroglial cultures derived from total cerebral hemispheres, cerebral cortex and striatum. Interestingly, these cultures expressed a low, but detectable, level of mGluR5 mRNA. The more sensitive technique Reverse Transcription-Polymerase Chain Reaction (RT-PCR) confirmed the presence of mGluR5 transcript in cultured astrocytes and, in addition, revealed the presence of mGluR3 mRNA. The lack of expression of mGluR5 in CG-4 cells, a rat cell line able to differentiate in type-2 astrocytes or oligodendrocytes depending on the culture conditions, suggested that the presence of mGluR5 was not a general feature of cells of glial origin. Moreover, all the examined mGluR transcripts were undetectable by RT-PCR in CG4 cells. In order to confirm the possible expression of mGluR5 in cell of glial origin we examined the mRNA levels for this receptor in tissue samples from human gliomas obtained after surgical resection of the tumors: only 1 sample (grade II astrocytoma), out of 8 examined, showed the presence of mGluR5 mRNA. In conclusion our data show that the only cloned metabotropic receptor linked to phosphoinositide hydrolysis, whose expression is detectable in cultured type-1 astrocytes, is mGluR5. It remains to be established if the low level of expression of mGluR3 could be responsible for the group II metabotropic glutamate receptor activity previously observed in cultured astroglial cells.

AMPA receptor subunits are differentially expressed in parvalbumin- and calretinin-positive neurons of the rat hippocampus

Recent studies suggest a functional diversity of native α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate‐type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)‐ and calretinin (CR)‐positive cells, which represent different populations of non‐principal cells. To this end, non‐radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double‐immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV‐containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR‐A and GluR‐D mRNAs, moderate levels of GluR‐C and low levels of GluR‐B mRNAs in all the examined regions of hippocampus. The vast majority of CR‐containing cells have a much lower expression of GluR‐A, ‐C and ‐D mRNA than PV‐positive neurons, although similarly featuring low levels of GluR‐B mRNA. Only a subpopulation of CR‐containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR‐A and ‐D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR‐B subunit expression, PV‐ and CR‐containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.

Ischemia-induced changes in the immunoreactivity for endothelin and other vasoactive peptides in the brain of the Mongolian gerbil

Summary: Immunoreactivity for vasoactive peptides [endothelin (ET); calcitonin gene‐related peptide (CGRP); atrial natriuretic peptide (ANP); neuropeptide Y (NPY)] was investigated in nervous tissue of Mongolian gerbils in which the common carotid artery (CCA) was temporarily occluded (30 min‐4 h) on one side, provoking transient unilateral ischemia at the forebrain level. Observations were carried out in a group of animals that were perfused promptly after CCA reopening, and in a group of animals that were perfused 12 h later. In animals of the first group, darker immunostaining was usually observed for most peptides in the forebrain ipsilateral to the CCA occlusion. Computer‐assisted densitometric analysis showed that the asymmetry was relevant for ET, CGRP, and ANP, and almost undetectable for NPY. In animals of the second group, areas of tissue degeneration were observed. In these areas, ET immunoreactivity was markedly denser, whereas immunoreactivity for the remaining peptides was about at the background level. It is concluded that ischemia induces an increase in both vasoconstrictor and vasodilator peptides that in areas of moderate ischemia might maintain a residual tissue perfusion. In areas of severe hypoxia, a predominant ET‐induced vasoconstriction would contribute to tissue damage.

Influences of pyramidal tract on the subthalamic nucleus in the cat.

In adult cats, with mesencephalic decerebration sparing the cerebral peduncles and ablation of the sensorimotor cortex, changes in firing of single cells of subthalamic nucleus (STN) were analyzed upon stimulation of ipsilateral medullary pyramidal tract (PT). Twenty-two out of 44 of the STN cells exhibited, following PT stimulation, discharge changes that in the greatest part of cases (91%) were excitatory in nature. Excitations, always followed by inhibitory rebound, appeared with latency values compatible with a monosynaptic linkage.

Resveratrol inhibits transforming growth factor-β-induced proliferation and differentiation of ex vivo human lung fibroblasts into myofibroblasts through ERK/Akt inhibition and PTEN restoration.

ABSTRACT The authors investigated the role of resveratrol (RV), a natural poliphenolic molecule with several biological activities, in transforming growth factor-β (TGF-β)–induced proliferation and differentiation of ex vivo human pulmonary fibroblasts into myofibroblasts. The effects of RV treatment were evaluated by analyzing TGF-β–induced α-smooth muscle actin (α-SMA) expression and collagen production, as well as cell proliferation of both normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. Results demonstrate that RV inhibits TGF-β–induced cell proliferation of both normal and pathological lung fibroblasts, attenuates α-SMA expression at both the mRNA and protein levels, and also inhibits intracellular collagen deposition. In order to understand the molecular mechanisms, the authors also investigated the effects of RV treatment on signaling pathways involved in TGF-β–induced fibrosis. The authors show that RV inhibited TGF-β–induced phosphorylation of both extracellular signal-regulated kinases (ERK1/2) and the serine/threonine kinase, Akt. Moreover, RV treatment blocked the TGF-β–induced decrease in phosphatase and tensin homolog (PTEN) expression levels.

PI3K p110γ overexpression in idiopathic pulmonary fibrosis lung tissue and fibroblast cells: in vitro effects of its inhibition.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease whose molecular pathogenesis remains unclear. In a recent paper, we demonstrated a key role for the PI3K pathway in both proliferation and differentiation into myofibroblasts of normal human lung fibroblasts treated with TGF-β. In this research, we assessed the expression of class I PI3K p110 isoforms in IPF lung tissue as well as in tissue-derived fibroblast cell lines. Moreover, we investigated the in vitro effects of the selective inhibition of p110 isoforms on IPF fibroblast proliferation and fibrogenic activity. IHC was performed on normal and IPF lung tissue. Expression levels of PI3K p110 isoforms were evaluated by western blot and flow cytometry analysis. Fibroblast cell lines were established from both normal and IPF tissue and the effects of selective pharmacological inhibition as well as specific gene silencing by small interfering RNAs were studied in vitro. No significant differences between normal and IPF tissue/tissue-derived fibroblasts were observed for the expression of PI3K p110 α, β and δ isoforms whereas p110γ was more greatly expressed in both IPF lung homogenates and ex vivo fibroblast cell lines. Myofibroblasts and bronchiolar basal cells in IPF lungs exhibited strong immunoreactivity for p110γ. Positive staining for the markers of proliferation proliferating cell nuclear antigen and cyclin D1 was also shown in cells of fibrolastic foci. Furthermore, both p110γ pharmacological inhibition and gene silencing were able to significantly inhibit proliferation rate as well as α-SMA expression in IPF fibroblasts. Our data suggest that PI3K p110γ isoform may have an important role in the etio-pathology of IPF and can be a specific pharmacological target.

Influences of cerebral cortex and cerebellum on the red nucleus of the rat

The aim of the present work was to investigate the unitary responses of neurons belonging to the magnocellular and parvocellular division of the red nucleus (RN) to stimulation of efferents from motor cortex and cerebellum. In anesthetized rats spontaneous discharges of rubro-olivary (RO) and rubrospinal (RS) neurons were tested for stimulation of motor cortex (CX), pyramidal tract (PT), interpositus (IN) and dentate (DN) cerebellar nuclei. It has been observed that the majority of RO and RS neurons were influenced by stimulation of both IN and DN as well as by activation of CX and PT. These results indicate that (1) a segregation of cerebral and cerebellar afferents to RN of rat does not exist and (2) convergent responses from the same cerebral and cerebellar structures have been observed in a high number of both RS and RO neurons.

Single Muscle Organization of Interposito-rubral Projections*

SummaryIn unanesthetized neuraxis intact cats microstimulation of the interpositus nucleus (IN) which activated a single flexor or extensor muscle in limbs, was used to investigate changes of unitary discharges of rubrospinal (RST) cells. Recordings were made from sites the stimulation of which excited the same muscle activated by the IN (agonist cells), its antagonist (antagonist cells) or heteronymous muscles (heteronymous cells).Cats submitted to chronic cerebellar decortication, acute brachium conjunctivum (BC) section, acute prerubral hemidecerebration or chronic prerubral hemidecerebration and contralateral BC section, were used as controls.It was shown that agonist RST cells were monosynaptically fired from IN, while antagonist cells were inhibited and the heteronymous ones were not influenced.Cerebellar efferents within the BC mediate both excitatory and inhibitory effects, but cerebellar cortex and prerubral structures were not involved in their production.