Nanoparticles of 2-deoxy-d-glucose functionalized poly(ethylene glycol)-co-poly(trimethylene carbonate) for dual-targeted drug delivery in glioma treatment

Based on the facilitative glucose transporter (GLUT) over-expression on both blood–brain barrier (BBB) and glioma cells, 2-deoxy-d-glucose modified poly(ethylene glycol)-co-poly(trimethylene carbonate) nanoparticles (dGlu–NP) were developed as a potential dual-targeted drug delivery system for enhancing the BBB penetration via GLUT-mediated transcytosis and improving the drug accumulation in the glioma via GLUT-mediated endocytosis. In vitro physicochemical characterization of the dual-targeted nanoparticulate system presented satisfactory size of 71 nm with uniform distribution, high encapsulation efficiency and adequate loading capacity of paclitaxel (PTX). Compared with non-glucosylated nanoparticles (NP), a significantly higher amount of dGlu–NP was internalized by RG-2 glioma cells through caveolae-mediated and clathrin-mediated endocytosis. Both of the transport ratios across the in vitro BBB model and the cytotoxicity of RG-2 cells after crossing the BBB were significantly greater of dGlu–NP/PTX than that of NP/PTX. In vivo fluorescent image indicated that dGlu–NP had high specificity and efficiency in intracranial tumor accumulation. The anti-glioblastoma efficacy of dGlu–NP/PTX was significantly enhanced in comparison with that of Taxol and NP/PTX. Preliminary safety tests showed no acute toxicity to hematological system, liver, kidney, heart, lung and spleen in mice after intravenous administration at a dose of 100 mg/kg blank dGlu–NP per day for a week. Therefore, these results indicated that dGlu–NP developed in this study could be a potential dual-targeted vehicle for brain glioma therapy.     

Effects of acute exposure to aluminum on blood–brain barrier and the protection of zinc

Aluminum and zinc are two important trace elements in an organism. Although several studies have demonstrated their impacts on the intelligence, very little was known about their effects on the integrity of blood–brain barrier (BBB). To study the effects of aluminum and zinc on the permeability of BBB, different doses of aluminum and appropriate zinc were administered to rats. Evans blue was detected in brain to determine the permeability of BBB. The ultrastructure of BBB was observed under the transmission electron microscope. Immunohistochemistry and Western blot method were used to detect the expression of skeleton protein F-actin and tight junction protein occludin in brain capillary endothelium. The data indicated that compared with the control group, Evans blue in brains increased (P < 0.01), the ultrastructure of BBB changed and the expression of F-actin and occludin decreased (P < 0.01) in the aluminum-toxic group. Compared with the aluminum-toxic groups, the permeability of BBB to Evans blue decreased (P < 0.01), the damage of the BBB ultrastructure was attenuated and the expression of F-actin and occludin increased (P < 0.05) in the aluminum–zinc group. Our present studies suggest that aluminum increases the permeability of BBB by changing its ultrastructure and the expression of occludin and F-actin. Zinc can protect the integrity of BBB in juvenile rats that are exposed to aluminum and inhibit the decrease of tight junction protein occludin and F-actin expression in BBB.     

Increased delivery of TAT across an endothelial monolayer following ischemic injury

There is a great need for the development of vehicles capable of delivering therapeutic cargoes across the blood–brain barrier (BBB) and into brain cells. Cell-penetrating peptides (CPPs), such as TAT, present one such solution, and have been used successfully in vivo to deliver neuroprotective cargoes to the brain in models of stroke and seizure. However, a significant discrepancy exists in the literature, as other groups have not had the same success. One commonality between the successful studies is a compromised BBB. In this study, we hypothesized that ischemic injury increases the transport of TAT across an endothelial monolayer (comprised of bEnd.3 cells) in vitro and, consequently, increases TAT-mediated delivery into astrocytes on the other side. In the 24 h following in vitro ischemia (oxygen-glucose deprivation), transendothelial electrical resistance (TEER) significantly decreased, indicating disruption of BBB integrity. Concomitantly, the transport of a green fluorescent protein (GFP)-TAT fusion protein significantly increased, and the transduction of GFP-TAT into astrocytes cultured on the other side of the endothelial monolayer significantly increased. These results explain why TAT-mediated delivery of therapeutic cargoes is successful in the ischemic brain but not in the uninjured brain with an intact BBB, highlighting the necessity for continued development of delivery vehicles. We conclude that although TAT may not be an efficient vehicle for trans-BBB delivery across an intact BBB, it may have utility in clinical situations when the BBB is disrupted.     

Hydrogen-rich saline reduces oxidative stress and inflammation by inhibit of JNK and NF-κB activation in a rat model of amyloid-beta-induced Alzheimer's disease

This study is to examine if hydrogen-rich saline reduced amyloid-beta (Aβ) induced neural inflammation and oxidative stress in a rat model by attenuation of activation of JNK and NF-κB. Sprague–Dawley male rats (n = 18, 280–330 g) were divided into three groups, sham operated, Aβ1–42 injected and Aβ1–42 plus hydrogen-rich saline treated animals. Hydrogen-rich saline (5 ml/kg, i.p., daily) was injected for 10 days after intraventricular injection of Aβ1–42. The levels of IL-1β were assessed by ELISA analysis, 8-OH-dG by immunohistochemistry in the brain slides, and JNK and NF-κB by immunohistochemistry and western blotting. After Aβ1–42 injection, the level of IL-1β, 8-OH-dG, JNK and NF-κB all increased in brain tissues, while hydrogen-rich saline treatment decreased the level of IL-1β, 8-OH-dG and the activation of JNK and NF-κB. In conclusion, hydrogen-rich saline prevented Aβ-induced neuroinflammation and oxidative stress, possibly by attenuatation of activation of c-Jun NH₂-terminal kinase (JNK) and nuclear factor-κB (NF-κB) in this rat model.     

Electromechanical method coupling non-invasive skin impedance probing and in vivo subcutaneous liquid microinjection: controlling the diffusion pattern of nanoparticles within living soft tissues

Transdermal drug delivery is the way to transport drug carriers, such as nanoparticles, across the skin barrier to the dermal and/or subcutaneous layer. In order to control the transdermal drug delivery process, based on the heterogeneous and nonlinear structures of the skin tissues, we developed a novel electromechanical method combining in vivo local skin impedance probing, subcutaneous micro-injection of colloidal nanoparticles, and transcutaneous electrical stimulation. Experiments on the nude mice using in vivo fluorescence imaging exhibited significantly different apparent diffusion patterns of the nanoparticles depending on the skin impedance: Anisotropic and isotropic patterns were observed upon injection into low and high impedance points, respectively. This result implies that the physical complexity in living tissues may cause anisotropic diffusion of drug carriers, and can be used as a parameter for controlling drug delivery process. This method also can be combined with microneedle-based drug release systems, micro-fabricated needle-electrodes, and/or advanced in vivo targeting/imaging technologies using nanoparticles.     

Recombinant human erythropoietin prevents motor neuron apoptosis in a rat model of cervical sub-acute spinal cord compression

The objective of the study was to investigate the effects of recombinant human erythropoietin (rhEPO) in a rat model of cervical sub-acute spinal cord compression. 80 Wistar rats were randomly divided into 4 groups. Rats in the sham group (Group A, n = 5) underwent surgical procedures without cervical spinal cord compression; while rats in other groups were subjected to the spinal compression process. In the control group (Group B, n = 25), rats received an i.v. injection of 1 mL saline at day 7 post-surgery. Rats in the low-dose group (Group C, n = 25) and the high-dose group (Group D, n = 25) were treated with rhEPO at 500 units/kg body-weight and 5000 units/kg, respectively, via intravenous injection at day 7 post surgery. Limb motor function was scored by Basso–Beattie–Bresnahan (BBB) standards at 3, 7, 14, 21 and 28 days post-surgery. The distribution and quantities of EPO and its receptor (EPO-R) in the compressed segment of the spinal cord were detected by immunohistochemistry. Motor neuron apoptosis in the spinal cord was evaluated using TUNEL staining and flow cytometry at the indicated time points. Finally, IL-8, TNF-α, IL-6, and IL-1β levels in the compressed cervical spinal cord were determined by ELISA within the lesion epicenter at each time point post-surgery. The data suggest that expression of EPO-R was significantly increased following sub-acute cervical spinal cord compression; Groups C and D exhibited better BBB scores at all observed time points compared with the control group (p < 0.01). Using TUNEL staining and FCM, we observed that rhEPO profoundly inhibited motor neuron apoptosis in the spinal cord at day 21 (p < 0.01). Additionally, treatment with rhEPO halted the elevation of inflammatory cytokines. rhEPO administration decreased motor neuron apoptosis in the cervical spinal cord, improved motor functions and reduced the inflammatory response in a sub-acute cervical spinal cord compression model. Moreover, sustained treatment with low doses of rhEPO revealed a positive therapeutic effect.     

The brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(ε-caprolactone) nanoparticles

In order to evaluate the potential and mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(?-caprolactone)nanoparticles (ANG-PEG-NP) as brain targeting drug delivery system, Rhodamine B isothiocyanate (RBITC) was used as a fluorescent probe molecule to label ANG-PEG-NP through covalent bonding. The brain transcytosis across the blood-brain barrier (BBB) and brain delivery in mice of RBITC labeled ANG-PEG-NP were investigated in this paper. Results showed that ANG-PEG-NP enhanced significantly the uptake by BCECs compared with that of PEG-NP through caveolae- and clathrin-mediated endocytosis, involving a time-dependent, concentration-dependent and energy-dependent mode. The transport of ANG-PEG-NP across the in vitro BBB model was significantly increased than that of PEG-NP. After injection a dose of 100 mg/kg RBITC labeled ANG-PEG-NP or PEG-NP in mouse caudal vein, the brain coronal section showed a higher accumulation of ANG-PEG-NP in the cortical layer, lateral ventricle, third ventricles and hippocampus than that of PEG-NP. By using an excess of free LRP ligand (Angiopep-2 and/or Aprotinin) as a specific receptor inhibitor, it was evidenced that the uptake by BCECs in vitro, transport across in vitro BBB model and penetration into brain tissue in vivo of RBITC labeled ANG-PEG-NP could be inhibited significantly, which demonstrated the brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(?-caprolactone)nanoparticles might be a LRP receptor mediated transcytosis process. Understanding these issues is important for the future development of ANG-PEG-NP as a brain targeting drug delivery system for neurodegenerative disorders including glioma and Alzheimer’s disease.     

Angiotensin-converting enzyme (ACE) inhibitors exacerbate histological damage and motor deficits after experimental traumatic brain injury

Angiotensin-converting enzyme (ACE) inhibitors are widely used as blood pressure medications in hypertensive individuals. However, ACE inhibitors also play an integral role in the breakdown of neuronal substance P, which has been recently implicated in the development of functional deficits following traumatic brain injury (TBI). The present study therefore examined the effects of ACE inhibitors on histological and motor outcome following TBI. Male Sprague–Dawley rats were treated with Captopril, Enalapril or equal volume saline for 7 days prior to the induction of diffuse TBI using the impact acceleration model. At 5 h post-injury, animals administered Captopril demonstrated significantly increased substance P immunoreactivity compared to vehicle controls (p < 0.01), and increased dark cell change that persisted to 7 days post-trauma. Captopril also resulted in exacerbated motor deficits compared to vehicle treated animals (p < 0.05) as assessed by the rotarod test over a 7-day post-traumatic period. Administration of the alternative ACE inhibitor, Enalapril, likewise exacerbated motor deficits, confirming a class effect of ACE inhibitors rather than a compound effect specific to Captopril. We conclude that ACE inhibitors are deleterious to outcome following TBI, presumably by impairing the degradation of substance P and increasing substance P mediated neuronal injury.     

Effect of combined therapy with ephedrine and hyperbaric oxygen on neonatal hypoxic–ischemic brain injury

Perinatal hypoxic–ischemic (HI) is a major cause of brain injury in the newborn, and there is a lack of effective therapies to reduce injury-related disorders. The aim of the present study was to evaluate the effect of a combination of ephedrine and hyperbaric oxygen (HBO) on neonatal hypoxic–ischemic brain injury. 7-day-old Sprague–Dawley rat pups were randomly divided into sham operation, HI, ephedrine, HBO, and combined group. The ephedrine group was intraperitoneally injected with ephedrine, HBO group was treated for 2 h at 2.5 absolute atmosphere (ATA) per day, the combined group received both ephedrine and HBO treatments, the sham operation and HI groups were intraperitoneally injected with normal saline. Rat brains at 7 days after HI, were collected to determine histopathological damage and the expression levels of Caspase-3 and Nogo-A. Four weeks after insult, animals were challenged with Morris water maze test. The expressions of Caspase-3 and Nogo-A were reduced in treating groups compared to those in HI group (P < 0.01). Compared with the single treatment groups, the expression levels of Caspase-3 and Nogo-A were significantly reduced in the combined group (P < 0.01). Compared with the single treatment groups, the average time of escape latency was significantly shorter (P < 0.01) and the number of platform location crossing was more (P < 0.05) in combined group. These findings indicate that the combination of ephedrine and HBO can enhance the neuroprotective effect in the neonatal rat HI model partially mediated by inhibiting Caspase-3 and Nogo-A pathways.     

Effect of nitric oxide synthase inhibitor and NMDA receptor antagonist on the development of nicotine sensitization of nucleus accumbens dopamine release: An in vivo microdialysis study

We have previously found that the neuronal nitric oxide synthase inhibitor N-nitro-l-arginine (l-NNA) and the noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 prevent behavioral sensitization to nicotine. This study aimed to investigate the effect of l-NNA and MK-801 on a neurochemical component of nicotine sensitization by evaluating the effect of the drugs on nicotine sensitization of nucleus accumbens dopamine (DA) release. Sprague–Dawley rats were pretreated with l-NNA (15 mg/kg, i.p.), MK-801 (0.3 mg/kg, i.p.), or saline 30 min before injection of nicotine (0.4 mg/kg, s.c., once daily) for seven consecutive days. Twenty-four hours after the last drug injection, animals were challenged with local perfusion of 5 mM nicotine into the shell of nucleus accumbens for 60 min and DA release was monitored using in vivo microdialysis. In rats treated with repeated nicotine, acute nicotine challenge induced a greater increase of accumbal DA release than in saline-treated animals (maximal DA response = 969 ± 235% (mean ± S.E.M.) of basal level versus 520 ± 93%, p = 0.042). Co-administration of l-NNA or MK-801 with nicotine attenuated an increase of DA release elicited by acute nicotine challenge, compared with nicotine alone (maximal DA response = 293 ± 58% and 445 ± 90% of basal level, respectively versus 969 ± 235%, p = 0.004 and p = 0.013, respectively). These data demonstrate that l-NNA and MK-801 block the development of nicotine sensitization of nucleus accumbens DA release, further supporting the involvement of nitric oxide and NMDA receptors in the development of behavioral sensitization to nicotine.     

NOC-9, a selective nitric oxide donor,induces flight reactions in the dorsolateral periaqueductal gray of rats by activating soluble guanylate cyclase

Previous studies have showed that SIN-1, a nitric oxide (NO) donor, injected into the dorsolateral column of the periaqueductal gray (dlPAG) induces flight reactions. This drug, however, can also produce peroxynitrite, which may interfere in this effect. In addition, it is also unknown if this effect is mediated by local activation of soluble guanylate cyclase (sGC). The aims of this study, therefore, were (1) to investigate if NOC-9 (6-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine), a NO donor that does not produce peroxynitrite, would produce flight reactions after intra-dlPAG administration similar to those induced by SIN-1; (2) to verify if these responses could be prevented by local injection of a selective guanylate cyclase inhibitor (ODQ). Male Wistar rats (n = 5–12) with cannulae aimed at the dlPAG received injections of TRIS (pH 10.0, 0.5 μl), NOC-9 (75 and 150 nmol), saline or SIN-1 (200 nmol) and were placed in an open arena for 10 min. In a subsequent experiment animals (n = 7–8) were pretreated with ODQ (1 nmol/0.5 μl) before receiving NOC-9 150 nmol. NOC-9 induced a significant dose-dependent increase in flight reactions in the first minute after injection (% of animals displaying flight: vehicle = 0%, NOC 75 = 67%, NOC 150 = 75%). SIN-1 had a similar effect (100% of animals showing flight) but the effects lasted longer (10 min) than those of NOC-9. The effect of NOC-9 (150 nmol) was prevented by pretreatment with ODQ (% of animals displaying flight: vehicle + NOC 150 = 71%, ODQ + NOC 150 = 37%). The results suggest that NO donors injected into the dlPAG induce defensive responses that are not mediated by secondary peroxynitrite production. Moreover, they also indicate that these defensive responses depend on activation of local sGC. The data strengthen the proposal that NO can modulate defensive reactions in the dlPAG.     

Dual-functional nanoparticles targeting amyloid plaques in the brains of Alzheimer's disease mice

Alzheimer's disease (AD) is a common neurodegenerative disorder with few treatments. The limitations imposed by the blood–brain barrier (BBB) and the non-selective distribution of drugs in the brain have hindered the effective treatment of AD and may result in severe side effects on the normal brains. We developed a dual-functional nanoparticle drug delivery system based on a PEGylated poly (lactic acid) (PLA) polymer. Two targeting peptides that were screened by phage display, TGN and QSH, were conjugated to the surface of the nanoparticles. TGN specifically targets ligands at the BBB, while QSH has good affinity with Aβ_1-42, which is the main component of amyloid plaque. Tests probing the bEnd.3 cell uptake and in vivo imaging were conducted to determine the best density of TGN on the nanoparticles' surfaces. The optimal amount of QSH was studied using a Thioflavin T (ThT) binding assay and surface plasmon resonance (SPR) experiments. The optimal maleimide/peptide molar ratio was 3 for both TGN and QSH on the surface of the nanoparticles (T₃Q₃-NP), and these nanoparticles achieved enhanced and precise targeted delivery to amyloid plaque in the brains of AD model mice. A MTT assay also validated the safety of this dual-targeted delivery system; little cytotoxicity was demonstrated with both bEnd.3 and PC 12 cells. In conclusion, the T₃Q₃-NP might be a valuable targeting system for AD diagnosis and therapy.     

Blood brain barrier (BBB) dysfunction associated with increased expression of tissue and urokinase plasminogen activators following peripheral thermal injury

Emerging data suggests the serine proteases, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA), may play a detrimental role in traumatic states leading to compromise of the blood brain barrier (BBB). The purpose of our study was to define the role of endogenous tPA and uPA on the BBB following peripheral burn injuries. Adult male Sprague–Dawley rats (n = 46) were studied in control and thermal injury groups. Rats were anesthetized and submerged in 100 °C water for 6 s producing a third degree burn affecting 60–70% of the total body surface area. BBB dysfunction was then evaluated by measuring the amount of Evans blue and by calculating the water content in the brain. Levels of tPA and uPA mRNA in the brain were determined with real-time polymerase chain reaction (PCR) at 3 and 7 h post-injury. Results showed an increase in the brain water content and the presence of Evans blue in the brain tissue of thermally injured rats, temporally associated with an increased expression of endogenous tPA and uPA. Our study demonstrates that peripheral thermal injury does induce an increase in the permeability of the BBB. A possible mechanism may be an increased expression of tPA and uPA.     

Facilitated brain delivery of poly (ethylene glycol)–poly (lactic acid) nanoparticles by microbubble-enhanced unfocused ultrasound

Nanotechnology plays a unique instrumental role in the revolutionary development of brain-specific drug delivery, imaging, and diagnosis, but is highly limited by the existence of blood–brain barrier (BBB). In this study, microbubble-enhanced unfocused ultrasound (MEUUS) was developed as an approach to mediate an extensive brain delivery of poly (ethylene glycol) – poly (lactic acid) (PEG–PLA) nanoparticles. Following the MEUUS treatment, the nanoparticles signals were found to penetrate through the vascular walls and distributed deeply into the parenchyma at a significantly higher level (more than 250%) than those of the non-MEUUS treated control. Such effect was reversible and dependent on nanoparticles injection timing, sonication mode and mechanical index. Together with the transmission electron microscopy analysis, the increased brain accumulation of nanoparticles was claimed to be largely mediated by an ultrasound-induced stable cavitation of the microbubble which resulted in mechanical stretching of the vessel wall and consequently induced cellular transcytosis of the nanoparticles. The MEUUS technique was also used to facilitate the brain delivery of PEG–PLA nanoparticles functionalized with amyloid beta-specific antibody 6E10 for enabling the recognition of the hallmarks of Alzheimer's disease that widely distributed in the brain. No erythrocytes extravasation and other visible damages in the brain were detected following the MEUUS treatment. These findings together indicated that unfocused ultrasound with the aid of microbubble could effectively improve the brain delivery of nanoparticles, and this approach might serve as a safe and flexible platform for the potential application of nanoparticles in the diagnosis and therapy of brain diseases.     

Daytime modulation of cortical spreading depression according to blood glucose levels

The aim of this study was to investigate the effects of daytime and blood glucose levels on the propagation of cortical spreading depression (SD). Thirty-nine male Wistar rats were used. Animals were housed 5 per cage with a 12-h, light–dark cycle (lights on at 0600 h). Food and water were available ad libitum, and animals were fasted the night before the experiments. Cortical SD was recorded continuously for 3 h using Ag–AgCl agar-Ringer electrodes placed on the parietal cortex. Every 20 min, SD was elicited by 2% KCl stimulation of the frontal cortex for 1 min. After 1 h of SD-recording, blood glucose levels were measured, and animals were injected intravenously either with glucose (40% solution, 1 mL), insulin (0.3 U/100 g of body weight), or mannitol (20% solution, 1 mL). In the middle of the light period, which corresponds to zeitgeber time (ZT)5, 8 animals received glucose, 7 received insulin, and 6 received mannitol. In another experimental set, glucose or insulin was administered at ZT12 (at the end of the light period); 12 rats received glucose, and 6 received insulin. All the animals that received glucose were hyperglycaemic (P < 0.01), and the hyperglycaemia was less pronounced in the ZT12 group (P < 0.05; Student's t-test). Insulin induced acute hypoglycaemia in animals of both groups (ZT5, P < 0.02; ZT12, P < 0.05; Student's t-test). Glucose injection at ZT5 reduced SD, whereas the insulin ZT5 group showed increased SD propagation (ANOVA, P < 0.05 and 0.01, respectively). Neither glucose nor insulin injection changed SD velocity at ZT12. We concluded that blood glucose levels change the velocity of SD propagation and that these effects are influenced by the daytime. Dark periods seemed to produce a resistance to cortical SD propagation.     

Inactivation of the ventromedial prefrontal cortex mimics re-emergence of heroin seeking caused by heroin reconditioning

The purpose of this study was to investigate the role of the ventromedial prefrontal cortex (vmPFC) in the expression of extinguished heroin seeking as measured by conditioned place preference (CPP) in males Sprague–Dawley rats (n = 25). Heroin place conditioning (0.3 mg/kg SC × 4 sessions) was followed by a test of preference 24 h later, extinction (saline × 4 sessions), heroin reconditioning (saline or 1.0 mg/kg × 1 session), and a second test of place preference 24 h later. Fifteen minutes prior to this test, rats received intra-vmPFC infusions (bilateral, 0.5 μl/side) of a mixture of GABA_A (muscimol; 0.03 nmol) and a GABA_B (baclofen; 0.3 nmol) agonists, or vehicle. As expected on the basis of previous studies, reconditioning with heroin resulted in the re-emergence of a CPP. Importantly, inactivation of the vmPFC produced the same effect in animals that did not receive heroin on the session of reconditioning. These results indicate that the vmPFC modulates expression of extinguished heroin seeking and suggest that prefrontal inhibitory mechanisms are involved in relapse to drug seeking.     

EGFP–EGF1-conjugated nanoparticles for targeting both neovascular and glioma cells in therapy of brain glioma

As neovascular and glioma cells were closely associated and might be mutually promoted in glioma growth, a dual-targeting strategy targeting to both neovascular and glioma cells would be more promising as compared with those targeting one of them. In this study, we reported a drug delivery system where nanoparticles were decorated with EGFP–EGF1 (ENP), a fusion protein derived from factor VII with special affinity for tissue factor (TF) over-expressed in glioma tissues, to facilitate anti-glioma delivery of paclitaxel (PTX) by targeting both neovascular and glioma cells. In vitro protein binding assay demonstrated that EGFP–EGF1 bound well to C6 cells and perturbed human umbilical vein endothelial cells (HUVEC) with a concentration-dependent manner but not to unperturbed HUVEC. EGFP–EGF1–TF interaction significantly enhanced nanoparticles uptake by perturbed HUVEC and glioma C6 cells as well as nanoparticles penetration in C6 glioma spheroids, and thus improved the cytotoxicity of their payload in both monolayer cells and glioma spheroids models. In vivo imaging of glioma-bearing mice demonstrated the specific accumulation of ENP in glioma tissues. In vivo distribution of nanoparticles intuitively showed ENP mainly sited in both extravascular glioma cells and neovascular cells. Pharmacodynamic results revealed that PTX-loaded ENP (ENP–PTX) significantly prolonged the median survival time of glioma-bearing mice compared with that of any other group. TUNEL assay and H&E staining showed that ENP–PTX treatment induced significantly more cell apoptosis and tumor necrosis compared with other treatments. In conclusion, the results of this contribution demonstrated the great potential of EGFP–EGF1-functionalized nanoparticles for dual-targeting therapy of brain glioma.     

Glutamate receptor blockade in the rostral ventromedial medulla reduces the force of multisegmental motor responses to supramaximal noxious stimuli

The rostral ventromedial medulla (RVM) has been established as part of a descending pain-modulatory pathway. While the RVM has been shown to modulate homosegmental nociceptive reflexes such as tail flick or hindpaw withdrawal, it is not known what role the RVM plays in modulating the magnitude of multisegmental, organized motor responses elicited by noxious stimuli. Using local blockade of glutamate receptors with the non-specific glutamate receptor antagonist kynurenate (known to selectively block nociceptive facilitatory ON-cells), we tested the hypothesis that the RVM facilitates the magnitude of multi-limb movements elicited by intense noxious stimuli. In male Sprague–Dawley rats, we determined the minimum alveolar concentration (MAC) of isoflurane necessary to block multi-limb motor responses to noxious tail clamp. MAC was determined so that all animals were anesthetized at an equipotent isoflurane concentration (0.7 MAC). Supramaximal mechanical stimulation of the hindpaw or electrical stimulation of the tail elicited synchronous, repetitive movements in all four limbs that ceased upon, or shortly after (<5 s) termination of the stimulus. Kynurenate microinjection (2 nmol) into the RVM significantly attenuated, by 40–60%, the peak and integrated limb forces elicited by noxious mechanical stimulation of the hindpaw (p < 0.001; two-way ANOVA; n = 8) or electrical stimulation of the tail (peak force: p < 0.011, two-way ANOVA; n = 8), with significant recovery 40–60 min following injection. The results suggest that glutamatergic excitation of RVM neurons, presumably ON-cells, facilitates organized, multi-limb escape responses to intense noxious stimuli.     

All-trans retinoic acid enhances bystander effect of suicide-gene therapy against medulloblastomas

In our previous study we evaluated the antitumor effect of herpes simplex virus-thymidine kinase gene (HSV-tk) on human medulloblastomas (MBs) in a therapeutic delivery system using the immortalized neural stem cell (NSC) line C17.2. However, our findings indicated that the bystander effect between C17.2tk and Daoy MB cells was weak compared to the bystander effect between NSCtk and C6 glioma cells. Gap junction intercellular communication (GJIC) is the main mechanism mediating the bystander effect in HSV-tk gene therapy. All-trans retinoic acid (ATRA) has been shown to up-regulate the expression of Connexin43 and GJIC. In this study we investigated the synergistic effect of ATRA and HSV-tk gene therapy in the treatment of MBs. We found that the expression of Connexin43 in Daoy cells was significantly increased when cells were exposed to 3 μmol/l of ATRA (P < 0.05). After co-culturing C17.2tk cells with Daoy cells at different ratios ranging from 1:1 to 1:16, ATRA significantly increased the bystander anti-tumor effect compared to ATRA-untreated cells (P < 0.05). In intracranial co-implantation experiments, mice co-implanted with C17.2tk/Daoy cells and treated with a combination of ATRA and GCV had significantly smaller tumors compared to the animals treated with GCV alone (P < 0.05). Together, our results show that ATRA enhanced the tumoricidal effect in HSVtk/GCV suicide gene therapy against Daoy MB cells by strengthening the bystander effect in vitro and in vivo.     

Memantine treatment decreases levels of secreted Alzheimer's amyloid precursor protein (APP) and amyloid beta (Aβ) peptide in the human neuroblastoma cells

Memantine, an uncompetitive NMDA receptor antagonist, is a FDA-approved drug used for the treatment of moderate-to-severe Alzheimer's disease (AD). Several studies have documented protective roles of memantine against amyloid beta (Aβ) peptide-mediated damage to neurons in both in vitro and in vivo models. Memantine is also effective in reducing amyloid burden in the brain of APP transgenic mice. However, the exact mechanism by which memantine provides protection against Aβ-mediated neurodegenerative cascade, including APP metabolism, remains to be elucidated. Herein, we investigated the effect of memantine on levels of the secreted form of Aβ precursor protein (APP), secreted Aβ and cell viability markers under short/acute conditions. We treated neuronal SK-N-SH cells with 10 μM memantine and measured levels of secreted total APP (sAPP), APPα isoform and Aβ_(1–40) in a time dependent manner for up to 24 h. Memantine significantly decreased the levels of the secreted form of sAPP, sAPPα and Aβ_(1–40) compared to vehicle treated cells. This change started as early as 8 h and continued for up to 24 h of drug treatment. Unlike sAPP, a slight non-significant increase in total intracellular APP level was observed in 24-h treated memantine cells. Taken together, these results suggest a role for memantine in the transport or trafficking of APP molecules away from the site of their proteolytic cleavage by the secretase enzymes. Such a novel property of memantine warrants further study to define its therapeutic utility.