Neurostatin and other O-acetylated gangliosides show anti-neuroinflammatory activity involving the NFκB pathway.

In many neuropathologies activated microglia and macrophages cause neurotoxicity and prolong the inflammatory response. We have previously characterized the glycosphingolipid Neurostatin (Nst), which potentially reduces these detrimental mechanisms. Nst, isolated from mammalian brain, is the GD1b ganglioside with O-acetylation of the outer sialic acid residue. Using the enzyme sialate-O-acetyltransferase (SOAT), we obtained several O-acetylated gangliosides and O-propionylated GD1b (PrGD1b). In the present study we investigated the anti-inflammatory effects of these compounds. Nst and other O-acetylated gangliosides reduced nitrite production in microglial cells which were activated with lipopolysaccharide (LPS), but did not affect nitrite production after their stimulation with interferon gamma (IFNγ). Structure-activity relationship analysis showed that Nst was the most active ganglioside as inhibitor of nitrite production. Its ceramide moiety is essential for this, and both, the O-acetylation and the monosaccharide chain are important for the anti-inflammatory activity of the gangliosides. We also found that Nst reduced iNOS, IL-6 and IL-12 transcription in LPS-induced microglia, likely by inhibiting nuclear localization of NFκB. In co-cultures, Nst reduced neuronal cell death caused by LPS-activated microglia. In vivo, Nst diminished microglia activation in a mouse model of acute neuroinflammation. We propose that Nst and other O-acetylated gangliosides are neuroprotective regulators of microglia activity under both physiological and pathological conditions.

Micellar Iron Oxide Nanoparticles Coated with Anti-Tumor Glycosides.

The synthesis procedure of nanoparticles based on thermal degradation produces organic solvent dispersible iron oxide nanoparticles (OA-IONP) with oleic acid coating and unique physicochemical properties of the core. Some glycosides with hydrophilic sugar moieties bound to oleyl hydrophobic chains have antimitotic activity on cancer cells but reduced in vivo applications because of the intrinsic low solubility in physiological media, and are prone to enzymatic hydrolysis. In this manuscript, we have synthetized and characterized OA-IONP-based micelles encapsulated within amphiphilic bioactive glycosides. The glycoside-coated IONP micelles were tested as Magnetic Resonance Imaging (MRI) contrast agents as well as antimitotics on rat glioma (C6) and human lung carcinoma (A549) cell lines. Micelle antimitotic activity was compared with the activity of the corresponding free glycosides. In general, all OA-IONP-based micellar formulations of these glycosides maintained their anti-tumor effects, and, in one case, showed an unusual therapeutic improvement. Finally, the micelles presented optimal relaxometric properties for their use as T2-weighed MRI contrast agents. Our results suggest that these bioactive hydrophilic nano-formulations are theranostic agents with synergistic properties obtained from two entities, which separately are not ready for in vivo applications, and strengthen the possibility of using biomolecules as both a coating for OA-IONP micellar stabilization and as drugs for therapy.

Integrated Stress Response as a Therapeutic Target for CNS Injuries.

Central nervous system (CNS) injuries, caused by cerebrovascular pathologies or mechanical contusions (e.g., traumatic brain injury, TBI) comprise a diverse group of disorders that share the activation of the integrated stress response (ISR). This pathway is an innate protective mechanism, with encouraging potential as therapeutic target for CNS injury repair. In this review, we will focus on the progress in understanding the role of the ISR and we will discuss the effects of various small molecules that target the ISR on different animal models of CNS injury.

Neuroprotection and Blood-Brain Barrier Restoration by Salubrinal After a Cortical Stab Injury.

Following a central nervous system (CNS) injury, restoration of the blood-brain barrier (BBB) integrity is essential for recovering homeostasis. When this process is delayed or impeded, blood substances and cells enter the CNS parenchyma, initiating an additional inflammatory process that extends the initial injury and causes so-called secondary neuronal loss. Astrocytes and profibrotic mesenchymal cells react to the injury and migrate to the lesion site, creating a new glia limitans that restores the BBB. This process is beneficial for the resolution of the inflammation, neuronal survival, and the initiation of the healing process. Salubrinal is a small molecule with neuroprotective properties in different animal models of stroke and trauma to the CNS. Here, we show that salubrinal increased neuronal survival in the neighbourhood of a cerebral cortex stab injury. Moreover, salubrinal reduced cortical blood leakage into the parenchyma of injured animals compared with injured controls. Adjacent to the site of injury, salubrinal induced immunoreactivity for platelet-derived growth factor subunit B (PDGF-B), a specific mitogenic factor for mesenchymal cells. This effect might be responsible for the increased immunoreactivity for fibronectin and the decreased activation of microglia and macrophages in injured mice treated with salubrinal, compared with injured controls. The immunoreactivity for PDGF-B colocalized with neuronal nuclei (NeuN), suggesting that cortical neurons in the proximity of the injury were the main source of PDGF-B. Our results suggest that after an injury, neurons play an important role in both, the healing process and the restoration of the BBB integrity. J. Cell. Physiol. 232: 1501-1510, 2017. © 2016 Wiley Periodicals, Inc.

TGFß Contributes to the Anti-inflammatory Effects of Tauroursodeoxycholic Acid on an Animal Model of Acute Neuroinflammation.

The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is a neuroprotective agent in various animal models of neuropathologies. We have previously shown the anti-inflammatory properties of TUDCA in an animal model of acute neuroinflammation. Here, we present a new anti-inflammatory mechanism of TUDCA through the regulation of transforming growth factor ß (TGFß) pathway. The bacterial lipopolysaccharide (LPS) was injected intravenously (iv) on TGFß reporter mice (Smad-binding element (SBE)/Tk-Luc) to study in their brains the real-time activation profile of the TGFß pathway in a non-invasive way. The activation of the TGFß pathway in the brain of SBE/Tk-Luc mice increased 24 h after LPS injection, compared to control animals. This activation peak increased further in mice treated with both LPS and TUDCA than in mice treated with LPS only. The enhanced TGFß activation in mice treated with LPS and TUDCA correlated with both an increase in TGFß3 transcript in mouse brain and an increase in TGFß3 immunoreactivity in microglia/macrophages, endothelial cells, and neurons. Inhibition of the TGFß receptor with SB431542 drug reverted the effect of TUDCA on microglia/macrophages activation and on TGFß3 immunoreactivity. Under inflammatory conditions, treatment with TUDCA enhanced further the activation of TGFß pathway in mouse brain and increased the expression of TGFß3. Therefore, the induction of TGFß3 by TUDCA might act as a positive feedback, increasing the initial activation of the TGFß pathway by the inflammatory stimulus. Our findings provide proof-of-concept that TGFß contributes to the anti-inflammatory effect of TUDCA under neuroinflammatory conditions.

TUDCA: An Agonist of the Bile Acid Receptor GPBAR1/TGR5 With Anti-Inflammatory Effects in Microglial Cells.

Bile acids are steroid acids found in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is neuroprotective in different animal models of stroke and neurological diseases. We have previously shown that TUDCA has anti-inflammatory effects on glial cell cultures and in a mouse model of acute neuroinflammation. We show now that microglial cells (central nervous system resident macrophages) express the G protein-coupled bile acid receptor 1/Takeda G protein-coupled receptor 5 (GPBAR1/TGR5) in vivo and in vitro. TUDCA binding to GPBAR1/TGR5 caused an increase in intracellular cAMP levels in microglia that induced anti-inflammatory markers, while reducing pro-inflammatory ones. This anti-inflammatory effect of TUDCA was inhibited by small interference RNA for GPBAR1/TGR5 receptor, as well as by treatment with a protein kinase A (PKA) inhibitor. In the mouse model of acute neuroinflammation, treating the animals with TUDCA was clearly anti-inflammatory. TUDCA biased the microglial phenotype in vivo and in vitro toward the anti-inflammatory. The bile acid receptor GPBAR1/TGR5 could be a new therapeutic target for pathologies coursing with neuroinflammation and microglia activation, such as traumatic brain injuries, stroke, or neurodegenerative diseases. TUDCA and other GPBAR1/TGR5 agonists need to be further investigated, to determine their potential in attenuating the neuropathologies associated with microglia activation. J. Cell. Physiol. 232: 2231-2245, 2017. © 2016 Wiley Periodicals, Inc.

Aldynoglia cells and modulation of RhoGTPase activity as useful tools for spinal cord injury repair.

A combined approach in spinal cord injury (SCI) therapy is the modulation of the cellular and molecular processes involved in glial scarring. Aldaynoglial cells are neural cell precursors with a high capacity to differentiate into neurons, promote axonal growth, wrapping and myelination of resident neurons. These important characteristics of aldaynoglia can be combined with specific inhibition of the RhoGTPase activity in astroglia and microglia that cause reduction of glial proliferation, retraction of glial cell processes and myelin production by oligodendrocytes. Previously we used experimental central nervous system (CNS) injury models, like spinal cord contusion and striatal lacunar infarction and observed that administration of RhoGTPase glycolipid inhibitor or aldaynoglial cells, respectively, produced a significant gain of functional recovery in treated animals. The combined therapy with neuro-regenerative properties strategy is highly desirable to treat SCI for functional potentiation of neurons and oligodendrocytes, resulting in better locomotor recovery. Here we suggest that treatment of spinal lesions with aldaynoglia from neurospheres plus local administration of a RhoGTPase inhibitor could have an additive effect and promote recovery from SCI.

Inhibition of glial proliferation, promotion of axonal growth and myelin production by synthetic glycolipid: A new approach for spinal cord injury treatment.

PURPOSE: After spinal cord injury (SCI) a glial scar is generated in the area affected that forms a barrier for axon growth and myelination, preventing functional recovery. Recently, we have described a synthetic glycolipid (IG20) that inhibited proliferation of human glioma cells. We show now that IG20 inhibited the proliferation of astrocytes and microglial cells, the principal cellular components of the glial scar, and promoting axonal outgrowth and myelin production in vitro. METHODS: Glial cells were inhibited with IG20 (IC50≈10 µM) and studied by RT-PCR, Western Blotting, immunoprecipitation and fluorescence microscopy. Axonal outgrowth in dorsal root ganglia (DRG) and myelin production by oligodendrocytes were analyzed by immunocytochemistry. Adult rats were assayed in spinal cord contusion model and the recovery of treated animals (n = 6) and controls (n = 6) was followed. RESULTS: The IG20 was localized in the cytosol of glial cells, forming a complex with RhoGDIα, a regulator of RhoGTPases. Treatment of astroglial cultures with IG20 increase the expression of BDNF receptor genes (TrkBT1, TrkB Full). IG20 reduced the astroglial marker GFAP, while increasing production of myelin basic protein in oligodendrocytes and promoted axonal outgrowth from DRG neurons. Local injection of IG20, near a spinal cord contusion, promoted the recovery of lesioned animals analyzed by BBB test (P <  0.05). CONCLUSIONS: We propose that inhibition of astrocytes and microglia by IG20 could be diminished the glial scar formation, inducing the re-growth and myelination of axons, these elements constitute a new approach for SCI therapy.

New oleyl glycoside as anti-cancer agent that targets on neutral sphingomyelinase.

We designed and synthesized two anomeric oleyl glucosaminides as anti-cancer agents where the presence of a trifluoroacetyl group close to the anomeric center makes them resistant to hydrolysis by hexosaminidases. The oleyl glycosides share key structural features with synthetic and natural oleyl derivatives that have been reported to exhibit anti-cancer properties. While both glycosides showed antiproliferative activity on cancer cell lines, only the α-anomer caused endoplasmic reticulum (ER) stress and cell death on C6 glioma cells. Analysis of sphingolipids and glycosphingolipds in cells treated with the glycosides showed that the α-anomer caused a drastic accumulation of ceramide and glucosylceramide and reduction of lactosylceramide and GM3 ganglioside at concentrations above a threshold of 20 µM. In order to understand how ceramide levels increase in response to α-glycoside treatment, further investigations were done using specific inhibitors of sphingolipid metabolic pathways. The pretreatment with 3-O-methylsphingomyelin (a neutral sphingomyelinase inhibitor) restored sphingomyelin levels together with the lactosylceramide and GM3 ganglioside levels and prevented the ER stress and cell death caused by the α-glycoside. The results indicated that the activation of neutral sphingomyelinase is the main cause of the alterations in sphingolipids that eventually lead to cell death. The new oleyl glycoside targets a key enzyme in sphingolipid metabolism with potential applications in cancer therapy.

Salubrinal inhibits the expression of proteoglycans and favors neurite outgrowth from cortical neurons in vitro.

After CNS injury, astrocytes and mesenchymal cells attempt to restore the disrupted glia limitans by secreting proteoglycans and extracellular matrix proteins (ECMs), forming the so-called glial scar. Although the glial scar is important in sealing the lesion, it is also a physical and functional barrier that prevents axonal regeneration. The synthesis of secretory proteins in the RER is under the control of the initiation factor of translation eIF2α. Inhibiting the synthesis of secretory proteins by increasing the phosphorylation of eIF2α, might be a pharmacologically efficient way of reducing proteoglycans and other profibrotic proteins present in the glial scar. Salubrinal, a neuroprotective drug, decreased the expression and secretion of proteoglycans and other profibrotic proteins induced by EGF or TGFß, maintaining eIF2α phosphorylated. Besides, Salubrinal also reduced the transcription of proteoglycans and other profibrotic proteins, suggesting that it induced the degradation of non-translated mRNA. In a model in vitro of the glial scar, cortical neurons grown on cocultures of astrocytes and fibroblasts with TGFß treated with Salubrinal, showed increased neurite outgrowth compared to untreated cells. Our results suggest that Salubrinal may be considered of therapeutic value facilitating axonal regeneration, by reducing overproduction and secretion of proteoglycans and profibrotic protein inhibitors of axonal growth.

Tauroursodeoxycholic acid reduces glial cell activation in an animal model of acute neuroinflammation.

BACKGROUND: Bile acids are steroid acids found predominantly in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is a neuroprotective agent in different animal models of stroke and neurological diseases. However, the anti-inflammatory properties of TUDCA in the central nervous system (CNS) remain unknown. METHODS: The acute neuroinflammation model of intracerebroventricular (icv) injection with bacterial lipopolysaccharide (LPS) in C57BL/6 adult mice was used herein. Immunoreactivity against Iba-1, GFAP, and VCAM-1 was measured in coronal sections in the mice hippocampus. Primary cultures of microglial cells and astrocytes were obtained from neonatal Wistar rats. Glial cells were treated with proinflammatory stimuli to determine the effect of TUDCA on nitrite production and activation of inducible enzyme nitric oxide synthase (iNOS) and NFκB luciferase reporters. We studied the effect of TUDCA on transcriptional induction of iNOS and monocyte chemotactic protein-1 (MCP-1) mRNA as well as induction of protein expression and phosphorylation of different proteins from the NFκB pathway. RESULTS: TUDCA specifically reduces microglial reactivity in the hippocampus of mice treated by icv injection of LPS. TUDCA treatment reduced the production of nitrites by microglial cells and astrocytes induced by proinflammatory stimuli that led to transcriptional and translational diminution of the iNOS. This effect might be due to inhibition of the NFκB pathway, activated by proinflammatory stimuli. TUDCA decreased in vitro microglial migration induced by both IFN-γ and astrocytes treated with LPS plus IFN-γ. TUDCA inhibition of MCP-1 expression induced by proinflammatory stimuli could be in part responsible for this effect. VCAM-1 inmunoreactivity in the hippocampus of animals treated by icv LPS was reduced by TUDCA treatment, compared to animals treated with LPS alone. CONCLUSIONS: We show a triple anti-inflammatory effect of TUDCA on glial cells: i) reduced glial cell activation, ii) reduced microglial cell migratory capacity, and iii) reduced expression of chemoattractants (e.g., MCP-1) and vascular adhesion proteins (e.g., VCAM-1) required for microglial migration and blood monocyte invasion to the CNS inflammation site. Our results present a novel TUDCA anti-inflammatory mechanism, with therapeutic implications for inflammatory CNS diseases.

The effect of antitumor glycosides on glioma cells and tissues as studied by proton HR-MAS NMR spectroscopy.

The effect of the treatment with glycolipid derivatives on the metabolic profile of intact glioma cells and tumor tissues, investigated using proton high resolution magic angle spinning ((1)H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy, is reported here. Two compounds were used, a glycoside and its thioglycoside analogue, both showing anti-proliferative activity on glioma C6 cell cultures; however, only the thioglycoside exhibited antitumor activity in vivo. At the drug concentrations showing anti-proliferative activity in cell culture (20 and 40 µM), significant increases in choline containing metabolites were observed in the (1)H NMR spectra of the same intact cells. In vivo experiments in nude mice bearing tumors derived from implanted C6 glioma cells, showed that reduction of tumor volume was associated with significant changes in the metabolic profile of the same intact tumor tissues; and were similar to those observed in cell culture. Specifically, the activity of the compounds is mainly associated with an increase in choline and phosphocholine, in both the cell cultures and tumoral tissues. Taurine, a metabolite that has been considered a biomarker of apoptosis, correlated with the reduction of tumor volume. Thus, the results indicate that the mode of action of the glycoside involves, at least in part, alteration of phospholipid metabolism, resulting in cell death.

Agar-based bridges as biocompatible candidates to provide guide cues in spinal cord injury repair.

BACKGROUND: Spinal bridge implants are strategic to provide growth surfaces for axonal regeneration after spinal cord injuries. The design of an appropriate substrate, one that is suitable for implantation, must involve careful testing of the biomaterial properties both in vitro and in vivo. OBJECTIVE: The goal of this work was to test the structure, stability and biological response after spinal bridges implantation of several biopolymers, composed of mixtures of agar (AG), as structural matrix scaffold, with κ-carrageenan (Kc), gelatin (G), xanthan gum (Xn) and polysulfone (PS). METHODS: Biopolymer structures were studied by environmental scanning electron microscopy, whereas the stability of gels was analyzed by in vitro degradation and swelling tests. The biocompatibility of these materials and their ability to promote cell growth and axonal regeneration were studied by implantation of spinal bridges containing empty linear channels in an acute rat spinal cord transection model at thoracic level (T8). RESULTS AND CONCLUSIONS: All gel mixtures gave rise to porous structures and they were stables to degradation, excepting the AG+G mixture. Spinal bridges constructed from all mixtures were implanted during a month in adult rats. After this time a low host reaction occurred to all bridge materials as well as neurite and cell ingrowths through the empty channels. Neurites within the bridges were mostly peripheral sensory fibers such as those positive for CGRP, whereas there was a lack of regeneration of central axons crossing from the spinal tissue to bridges. Many of these neurites established closed contacts with non-myelin Schwann cells. The histological analysis revealed a high accumulation of collagen fibers within the channels. Unexpected was the apparent loss of channels linearity which affected the growth of neurites and cells, indicating the need for additional regeneration strategies and vertebrae bridge fixing.

Influence of chitosan concentration on cell viability and proliferation in vitro by changing film topography.

PURPOSE: Chitosan is a natural polysaccharide which can form gels and scaffolds that support its use as a biomaterial in various tissue engineering applications. A useful feature of chitosan polymer is that you can manipulate its properties easily. Thus, in this work we studied the effect of varying chitosan concentration in the topography and the biological properties of the chitosan films, as well as the effects in the structure of 3D gels in order to be used as nerve bridges. METHODS: Analysis of film topographies were addressed by swelling test and atomic force microscopy (AFM). In vitro biological properties were assessed through MTT viability assays on cultures of blood-brain barrier forming endothelial (bEnd5), glioma (C6) and postmitotic neuron (NGF-differentiated PC12) cell lines. The structure of tridimensional gels was studied by environmental scanning electron microscopy.
 RESULTS: Topography of 1% chitosan films showed a AFM profile with higher nano-roughness profile than that observed in 2% films, which was smoother. Moreover, swelling rate was not affected. Topography changes affected cell viability as shown by the MTT assays. Our results showed that 2% chitosan films promoted higher proliferation and viability of C6 and PC12 respectively than 1% films. Conversely, neither 1% nor 2% films promoted viability of bEnd5 cells. In order to establish the
feasibility of both type of chitosan solutions as nerve bridges, we constructed 3D gels by alkaline precipitation. Resulting gels showed that only 2% gels were rigid enough to be effectively used as nerve bridges. CONCLUSIONS: These results establish that changes in chitosan concentration affects the polymer surface topography, which has a direct effect in the growing cell behavior. Additionally, higher concentration of chitosan gels are required to be used in neural tissue engineering.

Synthetic glycolipids for glioma growth inhibition developed from neurostatin and NF115 compound.

Neurostatin, a natural glycosphingolipid, and NF115, a synthetic glycolipid, are inhibitors of glioma growth. While neurostatin shows high inhibitory activity on gliomas its abundance is low in mammalian brain. On the contrary NF115 exhibits less inhibitory activity on gliomas, but could be prepared by chemical synthesis. In this study we describe synthetic compounds, structurally related to NF115, capable of inhibiting glioma growth at low micromolar range. We used DNA microarray technology to compare the genes inhibited in U373-MG human glioma cells after treatment with the natural or synthetic inhibitor. New synthetic compounds were developed to interact with the product of Rho GDP dissociation inhibitor alpha gene, which was repressed in both treatments. Compounds that were inhibitors of glioma cell growth in assays for [3H]-thymidine incorporation were then injected in C6 tumor bearing rats and the tumor size in each animal group were measured. The GC-17, GC-4 and IG-5 are new compounds derived from NF115 and showed high antiproliferative activity on tumor cell lines. The GC-17 compound inhibited U373-MG glioblastoma cells (3.2 µM), the effects was fifty times more potent than NF115, and caused a significant reduction of tumor volume (P<0.05) when tested in Wistar rats allotransplanted with C6 glioma cells.

Specific synthesis of neurostatin and gangliosides O-acetylated in the outer sialic acids using a sialate transferase.

Gangliosides are sialic acid containing glycosphingolipids, commonly found on the outer leaflet of the plasma membrane. O-acetylation of sialic acid hydroxyl groups is one of the most common modifications in gangliosides. Studies on the biological activity of O-acetylated gangliosides have been limited by their scarcity in nature. This comparatively small change in ganglioside structure causes major changes in their physiological properties. When the ganglioside GD1b was O-acetylated in the outer sialic acid, it became the potent inhibitor of astroblast and astrocytoma proliferation called Neurostatin. Although various chemical and enzymatic methods to O-acetylate commercial gangliosides have been described, O-acetylation was nonspecific and produced many side-products that reduced the yield. An enzyme with O-acetyltransferase activity (SOAT) has been previously cloned from the bacteria Campylobacter jejuni. This enzyme catalyzed the acetylation of oligosaccharide-bound sialic acid, with high specificity for terminal alpha-2,8-linked residues. Using this enzyme and commercial gangliosides as starting material, we have specifically O-acetylated the gangliosides' outer sialic acids, to produce the corresponding gangliosides specifically O-acetylated in the sialic acid bound in alpha-2,3 and alpha-2,8 residues. We demonstrate here that O-acetylation occurred specifically in the C-9 position of the sialic acid. In summary, we present a new method of specific O-acetylation of ganglioside sialic acids that permits the large scale preparation of these modified glycosphingolipids, facilitating both, the study of their mechanism of antitumoral action and their use as therapeutic drugs for treating glioblastoma multiform (GBM) patients.

Neural differentiation of transplanted neural stem cells in a rat model of striatal lacunar infarction: light and electron microscopic observations.

The increased risk and prevalence of lacunar stroke and Parkinson's disease (PD) makes the search for better experimental models an important requirement for translational research. In this study we assess ischemic damage of the nigrostriatal pathway in a model of lacunar stroke evoked by damaging the perforating arteries in the territory of the substantia nigra (SN) of the rat after stereotaxic administration of endothelin-1 (ET-1), a potent vasoconstrictor peptide. We hypothesized that transplantation of neural stem cells (NSCs) with the capacity of differentiating into diverse cell types such as neurons and glia, but with limited proliferation potential, would constitute an alternative and/or adjuvant therapy for lacunar stroke. These cells showed neuritogenic activity in vitro and a high potential for neural differentiation. Light and electron microscopy immunocytochemistry was used to characterize GFP-positive neurons derived from the transplants. 48 h after ET-1 injection, we characterized an area of selective degeneration of dopaminergic neurons within the nigrostriatal pathway characterized with tissue necrosis and glial scar formation, with subsequent behavioral signs of Parkinsonism. Light microscopy showed that grafted cells within the striatal infarction zone differentiated with a high yield into mature glial cells (GFAP-positive) and neuron types present in the normal striatum. Electron microscopy revealed that NSCs-derived neurons integrated into the host circuitry establishing synaptic contacts, mostly of the asymmetric type. Astrocytes were closely associated with normal small-sized blood vessels in the area of infarct, suggesting a possible role in the regulation of the blood brain barrier and angiogenesis. Our results encourage the use of NSCs as a cell-replacement therapy for the treatment of human vascular Parkinsonism.

Cannabinoid agonist WIN 55,212-2 prevents the development of paclitaxel-induced peripheral neuropathy in rats. Possible involvement of spinal glial cells.

Spinal glial activation contributes to the development and maintenance of chronic pain states, including neuropathic pain of diverse etiologies. Cannabinoid compounds have shown antinociceptive properties in a variety of neuropathic pain models and are emerging as a promising class of drugs to treat neuropathic pain. Thus, the effects of repeated treatment with WIN 55,212-2, a synthetic cannabinoid agonist, were examined throughout the development of paclitaxel-induced peripheral neuropathy. Painful neuropathy was induced in male Wistar rats by intraperitoneal (i.p.) administration of paclitaxel (1mg/kg) on four alternate days. Paclitaxel-treated animals received WIN 55,212-2 (1mg/kg, i.p.) or minocycline (15 mg/kg, i.p.), a microglial inhibitor, daily for 14 days, simultaneous with the antineoplastic. The development of hypersensitive behaviors was assessed on days 1, 7, 14, 21 and 28 following the initial administration of drugs. Both the activation of glial cells (microglia and astrocytes) at day 29 and the time course of proinflammatory cytokine release within the spinal cord were also determined. Similar to minocycline, repeated administration of WIN 55,212-2 prevented the development of thermal hyperalgesia and mechanical allodynia in paclitaxel-treated rats. WIN 55,212-2 treatment also prevented spinal microglial and astrocytic activation evoked by paclitaxel at day 29 and attenuated the early production of spinal proinflammatory cytokines (interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α). Our results confirm changes in the reactivity of glial cells during the development of peripheral neuropathy induced by paclitaxel and support a preventive effect of WIN 55,212-2, probably via glial cells reactivity inactivation, on the development of this neuropathy.