Giovanna Traina

Lipid nanoparticles for brain targeting III. Long-term stability and in vivo toxicity

PURPOSE The aim of the work was to assess the long-term stability and the safety of lipid nanoparticles intended for brain drug delivery. METHODS Lipid nanoparticles, prepared by high pressure homogenization, were stored at room temperature and 4°C and monitored for their mean hydrodynamic diameter and Gaussian distribution width over time. Cetylpalmitate and polysorbate(®) 80 chemical integrity were investigated by nuclear magnetic resonance on diagnostic signals. Nanoparticle toxicity was assessed in chicken embryos by chorioallantoic membrane assay and in rodents by brain histological evaluation. RESULTS Data showed nanoparticle stability at 4°C over a period of time of 4 years with only a limited particle size increase while at room temperature destabilization was observed after 9 months. Nuclear magnetic resonance investigation confirmed the absence (<5%) of chemical degradation of the lipid matrix and the surfactant after 4 years of storage at 4°C. Chorioallantoic membrane assay and rat brain histology showed the absence of acute toxicity corroborating previously published data. CONCLUSIONS Cetylpalmitate nanoparticle long-term physical and chemical stability, together with the in vivo safety, corroborate the existing evidences of the high value of colloidal lipids as parenteral formulations and carriers for brain drug delivery.

Sensitization and dishabituation of swim induction in the leech Hirudo medicinalis: role of serotonin and cyclic AMP

In this paper the role of serotonin (5HT) and cyclic AMP (cAMP) in sensitization and dishabituation of swim induction (SI) has been investigated in the leech Hirudo medicinalis. Electrical stimulation of the body wall evokes swimming activity with a constant latency. In animals with a disconnection between head ganglion and segmental ganglia, repetitive stimulation induces habituation of swimming whereas brushing on the dorsal skin provokes sensitization of a naïve response or dishabituation of a previously habituated response. Our findings indicate that 5HT is the neurotransmitter underlying both sensitization and dishabituation of SI. Injection of the 5HT receptor blocking agent methysergide impaires the onset of sensitization and dishabituation induced by brushing. Moreover, injection of 5HT mimics these forms of nonassociative learning, whereas injection of dopamine does not. Finally, the effects of 5HT are mediated by cAMP: (1) after injections of specific adenylate cyclase inhibitors such as MDL 12.330A or SQ22536, brushing becomes ineffective in facilitating the SI in either non-habituated or habituated animals. (2) 8Br-cAMP application mimics both sensitization and dishabituation of SI.

Seasonal variation of serotonin content and nonassociative learning of swim induction in the leech Hirudo medicinalis

SummaryIt is possible to obtain habituation of swim induction by stimulating the leech with repetitive light electrical trains. After obtaining this simple form of nonassociative learning, it is also possible to potentiate its response by a series of nociceptive skin brushings (dishabituation). Serotonin applied to the animal is the only neurotransmitter found to mimick dishabituation. We have observed that in the period April–June most animals did not exhibit potentiation of the swimming response after nociceptive stimulation while injection of serotonin mimicked dishabituation as in the animals treated in the period October–March. We have seen correlation between the changes in nonassociative learning and the seasonal variation of serotonin levels in segmental ganglia. This finding strengthens the hypothesis of serotonin as the neurotransmitter mediating dishabituation in swim induction of the leech.

Acetyl-l-carnitine up-regulates expression of voltage-dependent anion channel in the rat brain

Acetyl-L-carnitine (ALC) exerts unique neuroprotective, neuromodulatory, and neurotrophic properties, which play an important role in counteracting various pathological processes, and have antioxidative properties, protecting cells against lipid peroxidation. In this study, suppression subtractive hybridization (SSH) method was applied for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts after treatment of rats with ALC. The technique generates an equalized representation of differentially expressed genes irrespective of their relative abundance and it is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes that are regulated after ALC treatment. In the present paper, we report the identification of the gene of mitochondrial voltage-dependent anion channel (VDAC) protein which is positively modulated by the ALC treatment. VDAC is a small pore-forming protein of the mitochondrial outer membrane. It represents an interesting tool for Ca(2+) homeostasis, and it plays a central role in apoptosis. In addition, VDAC seems to have a relevant role in the synaptic plasticity.

Effects of Somatostatin on Intracellular Calcium Concentration in PC12 Cells

Abstract: Somatostatin (SS) is a neuropeptide that is distributed in various regions of the CNS, where it may act as a neurotransmitter or neuromodulator. SS produces multiple effects in the CNS through interactions with membrane receptors. In particular, SS inhibits various secretory responses in different cell types. In the present study, we have investigated the effects of exogenous application of SS on the intracellular free Ca2+ concentration ([Ca2+]i) in PC12 cells, a rat pheochromocytoma cell line. SS did reduce the magnitude of the secondary, maintained Ca2+ influx brought about by K+ depolarization. Similar effects were obtained with the application of SS analogues, such as d‐Trp8‐SS, d‐Trp8‐d‐Cys14‐SS, CGP‐23996, and SMS‐201995. In addition, treatment with cyclo‐SS, a SS antagonist, did not alter [Ca2+]i. Experiments with selective blockers of different voltage‐dependent Ca2+ channels, such as methoxyverapamil (D600) and Ω‐conotoxin GVIA, demonstrated that the effects of SS on [Ca2+]i were mediated by voltage‐dependent Ca2+ channels of the L type. Control experiments with a membrane potential indicator, i.e., the fluorescent dye bisoxonol, excluded that SS influenced the level of the membrane potential. SS effects on PC12 cells suggest the possibility that this neuropeptide plays a role in the modulation of cell functional activity by altering Ca2+ influx.

Antioxidative capacity of Lactobacillus fermentum LF31 evaluated in vitro by oxygen radical absorbance capacity assay.

OBJECTIVE The aim of this study was to evaluate the performance of the overall antioxidant of Lactobacillus fermentum LF31 bacterium with prebiotic supplement in human colon cultured cells. METHODS The antioxidant capability of L. fermentum LF31 has been assayed in vitro on human colon adenocarcinoma HT-29 cell line using the oxygen radical absorbance capacity method. RESULTS The analysis revealed that the interaction of probiotic strain cells supplemented with a prebiotic exerts a remarkable antioxidant capacity. CONCLUSION The L. fermentum used in the present study exhibited significant in vitro antioxidant capacity, increasing the total antioxidant potential.

Neurotoxic effects of caulerpenyne

1. In this paper the authors tested the effect of caulerpenyne (CYN), a sesquiterpene synthesized by the green alga Caulerpa taxifolia onto the central nervous system of the leech Hirudo medicinalis. Investigations have been performed with three different approaches: neuroethological, electrophysiological and neurochemical techniques. 2. CYN application mimics the effect of a nociceptive stimulation (brushing), eliciting a clear-cut potentiation of the animal swim response to the test stimulus (non associative learning process such as sensitization). This effect is similar to that one induced by the endogenous neurotransmitter serotonin (5HT). 3. CYN strongly reduces the after-hyperpolarization (AHP) recorded from T sensory neurons. This effect overlaps that one produced by 5HT, but it is not affected by the serotonergic antagonist methysergide. 4. The decrease of AHP amplitude due to CYN application is observed also in presence of apamin, a blocking agent of Ca++-dependent K+ channels, suggesting that CYN is acting through the inhibition of the Na+/K+ electrogenic pump. 5. The depression of the AHP driven by CYN is not prevented by application of MDL 12330A, an adenylate cyclase inhibitor. On the other hand MDL 12330A counteracts the reduction of AHP due to 5HT application. 6. Incubation of the leech central nervous system with CYN induces the phosphorylation of proteins of 29, 50, 66 and 100 kDa. This pattern of phosphorylation is similar to that one elicited by 5HT treatment. 7. The data demonstrate that CYN exerts remarkable effects on leech neurons by acting onto specific molecular targets such as the Na+/K+ ATPase. This effect may influence important neural integrative functions and may explain the sensitizing action produced by the toxin on swim induction. Finally, caulerpenyne does not act through the pathways involved in the 5HT action, and its effect is not mediated by the second messenger cyclic AMP. The mechanism of action of CYN are still under investigations.

Cytoprotective Effect of Acetyl-l-Carnitine Evidenced by Analysis of Gene Expression in the Rat Brain

Acetyl-l-carnitine (ALC), the acetyl ester of l-carnitine, is a naturally occurring substance that when administered at supraphysiological concentrations is neuroprotective. ALC plays an essential role in intermediary and mitochondrial metabolism. It has also neurotrophic and antioxidant actions. ALC has demonstrated efficacy and high tolerability in the treatment of neuropathies of various etiologies, and it is a molecule of considerable interest for its clinical application in various neural disorders, such as Alzheimer’s disease and painful neuropathies, although little is known regarding the effects of ALC on gene expression. Suppression subtractive hybridization methodology was used for the generation of subtracted complementary DNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after a chronic ALC treatment. In the present paper, we provide evidences for the up-regulation of the expression of prostaglandin D2 synthase, brain-specific Na+-dependent inorganic phosphate transporter, and cytochrome b oxidase, bc1 complex induced in the rat brain by ALC. On the contrary, ALC treatment down-regulates the expression of the gene of ferritin-H. Altogether, these results suggest that ALC might play a cytoprotective role against various brain stressors.

In the Rat Brain Acetyl-l-carnitine Treatment Modulates the Expression of Genes Involved in Neuronal Ceroid Lipofuscinosis

Acetyl-l-carnitine (ALC) is a naturally occurring substance that, when administered at supraphysiological concentration, is neuroprotective. It is a molecule of considerable interest for its clinical application in various neural disorders, including Alzheimer’s disease and painful neuropathies. Suppression subtractive hybridization methodology was used for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after ALC treatment. The method generates an equalized representation of differentially expressed genes irrespective of their relative abundance and it is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes which are regulated by ALC. We report that ALC treatment: (1) upregulates lysosomal H+/ATPase gene expression and (2) downregulates myelin basic protein gene expression. The expression of these genes is altered in some forms of neuronal ceroid lipofuscinosis (NCL) pathologies. In this case, ALC might rebalance the disorders underlying NCL disease represented by a disturbance in pH homeostasis affecting the acidification of vesicles transported to lysosomal compartment for degradation. This study provides evidence that ALC controls genes involved in these serious neurological pathologies and provides insights into the ways in which ALC might exert its therapeutic benefits.

Up-regulation of kinesin light-chain 1 gene expression by acetyl-l-carnitine: Therapeutic possibility in Alzheimer's disease

We investigated the effects of acetyl-l-carnitine on gene expression by means of the suppression subtractive hybridization method. The approach gives the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts after treatment of rats with acetyl-l-carnitine for 21 days. We observed that acetyl-l-carnitine increases the light-chain subunit of kinesin-1 gene expression. Recent evidences reported a link between kinesin-1 light-chain and Alzheimers disease. Pathological hallmarks of Alzheimers disease are potentially linked to alterations of the axonal compartments. Amyloid-beta peptide is a principal component of senile plaques and is considered to be central in the pathogenesis of the disease. The fast anterograde axonal transport of amyloid-beta peptide is mediated by direct binding to the light-chain subunit of kinesin-1. In this scenario, our results are of relevant importance for possible therapeutic intervention, suggesting a pathway for the treatment of Alzheimers disease.