Synthesis of Leucas Aspera Extract Loaded Gold-PLA-PEG-PLA Amphiphilic Copolymer Nanoconjugates: In Vitro Cytotoxicity and Anti-Inflammatory Activity Studies.

Gold nanoparticles (GNPs) are synthesized using the medicinal plant Leucas Aspera extract (LAE) and poly lactic acid-co-poly ethylene glycol-co-poly lactic acid (PLA-PEG-PLA) copolymer by water-in-oil (W/O) emulsion method. The proposed method of W/O emulsion technique involves synthesis of GNPs and loading of Leucas Aspera extract on to the PLA-PEG-PLA copolymer matrix simultaneously. The synthesized GNPs are characterized by Fourier transform infra-red (FTIR) spectroscopy, dynamic light scattering (DLS), X-ray diffractometry (XRD) and transmission electron microscopy (TEM). The GNPs-LAE loaded polymer NPs are examined for the in vitro cytotoxicity on South African green monkey's kidney cells. The GNPs-LAE loaded polymer nanoconjugates exhibit maximum up to 95% of cell viability with 100 µg concentration of GNPs in the sample. The GNPs-LAE loaded polymer NPs exhibit better anti-inflammatory activity when compared to the pure LAE.

In vitro drug release behavior, mechanism and antimicrobial activity of rifampicin loaded low molecular weight PLGA-PEG-PLGA triblock copolymeric nanospheres.

Poly (lactic-co-glycolic acid) (PLGA (92:8)) and a series of PLGA-PEG-PLGA tri block copolymers were synthesized by direct melt polycondensation. The copolymers were characterized by FTIR, and 1HNMR spectroscopic techniques, viscosity, gel permeation chromatography (GPC) and powder x-ray diffraction (XRD). The rifampicin (RIF) loaded polymeric nanospheres (NPs) were prepared by ultrasonication-W/O emulsification technique. The NPs have been characterized by field emission scanning electron microscopy (FESEM), TEM, powder X-ray diffraction (XRD), UVvisible spectroscopy and DLS measurements. The drug loaded triblock copolymeric NPs have five folds higher drug content and drug loading efficiency than that of PLGA microspheres (MPs). The in vitro drug release study shows that the drug loaded NPs showed an initial burst release after that sustained release up to 72 h. All the triblock copolymeric NPs follow anomalous drug diffusion mechanism while the PLGA MPs follow non-Fickian super case-II mechanism up to 12 h. The overall in-vitro release follows second order polynomial kinetics up to 72 h. The antimicrobial activity of the RIF loaded polymer NPs was compared with that of pure RIF and tetracycline (TA). The RIF loaded triblock copolymeric NPs inhibited the bacterial growth more effectively than the pure RIF and TA.

Asymmetrical changes of excitatory synaptic transmission in dopamine-denervated striatum after transient forebrain ischemia.

Spiny neurons in the neostriatum are highly vulnerable to cerebral ischemia. Recent studies have shown that the postischemic cell death in the right striatum was reduced after ipsilateral dopamine denervation whereas no protection was observed in the left striatum after dopamine denervation in the left side. In order to reveal the mechanisms of such asymmetrical protection, electrophysiological changes of dopamine-denervated striatal neurons were compared after ischemia between the left and right striatum using intracellular recording and staining techniques in vivo. No difference in cortically evoked initial excitatory postsynaptic potentials was found between the left and right striatum in intact animals after ipsilateral dopamine denervation. The initial excitatory postsynaptic potentials in the dopamine-denervated right striatum were suppressed after transient forebrain ischemia while no significant changes were found in the dopamine-denervated left striatum. Paired-pulse tests suggested that these changes involved presynaptic mechanisms. Although the incidence of a late depolarizing postsynaptic potential elicited by cortical stimulation increased after ischemia in both sides, the increase was greater in the left side. The analysis of current-voltage relationship of spiny neurons indicated that inward rectification in the left striatum transiently disappeared shortly after ischemia whereas that in the right side remained unchanged. The intrinsic excitability of spiny neurons in both sides were suppressed after ischemia, however, the suppression in the right side was stronger than in the left side. The above results demonstrate that after ipsilateral dopamine denervation, the depression of excitatory synaptic transmission and neuronal excitability in the right striatum is more severe than that in the left striatum following ischemia. The depression of excitatory synaptic transmission and neuronal excitability, therefore, might play an important role in neural protection after ischemic insult.

Enhanced excitatory synaptic transmission in spiny neurons of rat striatum after unilateral dopamine denervation.

The synaptic transmission and intrinsic membrane properties of spiny neurons in rat neostriatum were studied after unilateral dopamine depletion using in vivo intracellular recording and staining techniques. Two to four weeks after dopamine denervation, the spontaneous firing rate of spiny neurons increased and the spontaneous membrane potential fluctuation stayed at a more depolarized state for longer periods of time. The amplitude of cortically evoked initial excitatory postsynaptic potentials increased and a late excitatory postsynaptic potential that was occasionally found in control neurons was elicited from 23% of spiny neurons after dopamine denervation. No significant changes in intrinsic membrane properties of spiny neurons were observed after dopamine denervation. These results suggest that dopamine inhibits excitatory synaptic transmission of spiny neurons in naïve animals.

Differential changes of synaptic transmission in spiny neurons of rat neostriatum following transient forebrain ischemia.

Spiny neurons in neostriatum are vulnerable to cerebral ischemia. To reveal the mechanisms underlying the postischemic neuronal damage, the spontaneous activities, evoked postsynaptic potentials and membrane properties of spiny neurons in rat neostriatum were compared before and after transient forebrain ischemia using intracellular recording and staining techniques in vivo. In control animals the membrane properties of spiny neurons were about the same between the left and right neostriatum but the inhibitory synaptic transmission was stronger in the left striatum. After severe ischemia, the spontaneous firing and membrane potential fluctuation of spiny neurons dramatically reduced. The cortically evoked initial excitatory postsynaptic potentials were suppressed after ischemia indicated by the increase of stimulus threshold and the rise time of these components. The paired-pulse facilitation test indicated that such suppression might involve presynaptic mechanisms. The inhibitory postsynaptic potentials in spiny neurons were completely abolished after ischemia and never returned to the control levels. A late depolarizing postsynaptic potential that was elicited from approximately 5% of the control neurons by cortical stimulation could be evoked from approximately 30% of the neurons in the left striatum and approximately 50% in the right striatum after ischemia. The late depolarizing postsynaptic potential could not be induced after acute thalamic transection. The intrinsic excitability of spiny neurons was suppressed after ischemia evidenced by the significant increase of spike threshold and rheobase as well as the decrease of repetitive firing rate following ischemia. The membrane input resistance and time constant increased within 6 h following ischemia and the amplitude of fast afterhyperpolarization significantly increased after ischemia. These results indicate the depression of excitatory monosynaptic transmission, inhibitory synaptic transmission and excitability of spiny neurons after transient forebrain ischemia whereas the excitatory polysynaptic transmission in neostriatum was potentiated. The facilitation of excitatory polysynaptic transmission is stronger in the right neostriatum than in the left neostriatum after ischemia. The suppression of inhibitory component and the facilitation of excitatory polysynaptic transmission may contribute to the pathogenesis of neuronal injury in neostriatum after transient cerebral ischemia.

Desensitization of spinal 5-HT1A receptors to 8-OH-DPAT: an in vivo spinal reflex study.

Serotonergic influence on spinal monosynaptic transmission and the desensitization of spinal 5-HT1A receptors following a single pretreatment with a 5-HT1A ligand were examined in vivo in acutely spinalized adult rats. Administration of a selective 5-HT1A agonist, 8-OH-DPAT (0.1 mg kg-1) significantly depressed the monosynaptic mass reflex (MMR) amplitude, which was prevented effectively by S(-)-propranolol, a 5-HT1A antagonist. The inhibitory effect of 8-OH-DPAT on MMR amplitude was significantly attenuated with a single dose of 8-OH-DPAT (1 mg kg-1, s.c.) administered 24 h before the experiments, indicating a marked desensitization of spinal 5-HT1A receptors. Desensitization of 5-HT1A receptors could be reversed by treatment of spiperone (1 mg kg-1, i.p.) 3 h before 8-OH-DPAT pretreatment. These results demonstrate that 5-HT1A receptor functionally modulates the spinal motor output and confirms the ability of 8-OH-DPAT to desensitize presynaptic 5-HT1A receptors as observed for the first time in rat spinal cord.

Involvement of the presynaptic dopamine D2 receptor in the depression of spinal reflex by apomorphine.

The relative roles of D1 and D2 dopamine (DA) receptors in mediating apomorphine (APO)-induced changes in the spinal reflex was investigated. Low doses of APO, a DA receptor agonist (0.2 mg kg-1, i.v.), depressed the monosynaptic mass reflex (MMR) in spinalized rats. Pretreatment with the D2-specific antagonist, spiperone, 10 min before APO prevented the APO-induced MMR depression. Pretreatment with the D1 antagonist SCH 23390 failed to prevent the APO-induced depression. Interestingly, SCH 23390 pretreatment preferentially antagonized the depression induced by a high dose of APO (3 mg kg-1, i.v.). Pretreatment with SKF 38393, a selective D1 agonist, completely prevented the APO-induced MMR depression. These results suggest that inhibition of spinal transmission by low dose of APO may be mediated through its action on presynaptic D2 receptors and that D1 and D2 receptors are functionally coupled at the spinal level in modulating the spinal motor output.

Evidence for D1 dopamine receptor-mediated modulation of the synaptic transmission from motor axon collaterals to Renshaw cells in the rat spinal cord.

The possible modulatory role of D1 dopamine receptors on the excitability of lumbar spinal Renshaw cells was studied in anesthetized rats spinalized at T4 level. Burst responses elicited by single electrical shocks to ipsilateral ventral roots L6 (frequency 0.5 Hz, stimulus width 0.1 ms) and spontaneous activity were recorded extracellularly using conventional 3 M KCl filled glass micropipettes. The specific D1 agonist SKF 38393 (0.5-1 mg/kg i.v.) enhanced Renshaw cell burst responses by 20-60% (n = 7) and increased their spontaneous discharge rate (n = 3). This effect was clearly antagonized by the specific D1 antagonist SCH 23390 (1 mg/kg i.v.) although SCH 23390 proved ineffective per se. We conclude that SKF 38393 induced facilitation was due to activation of the specific D1 receptors which could be the functional counterpart of the presynaptic D2 receptors described earlier by us in the same synapse.