Yangzheng Feng

Influence of chronic morphine treatment on protein kinase C activity: comparison with butorphanol and implication for opioid tolerance

The aim of this study was to determine whether chronic opioid treatment could influence the protein kinase C (PKC) activity in the rat brain. Chronic morphine (microns agonist) but not butorphanol (mu/delta/kappa mixed agonist) treatment enhanced cytosolic PKC activity in the pons/medulla, but not in the cytosolic fractions of cortex and midbrain regions. Concomitant administration of the opioid receptor antagonist, naloxone, blocked the PKC upregulation by chronic morphine. Chronic administration of morphine and butorphanol produced no change in the membrane PKC activity. Antinociceptive tolerance to morphine but not to butorphanol was developed under these conditions. These results suggest that chronic morphine administration leads to an upregulation of the cytosolic PKC activity in the pons/medulla through repeated activation of mu opioid receptors and that the PKC upregulation in this specific area may contribute to the morphine tolerance.

Agmatine Suppresses Nitric Oxide Production and Attenuates Hypoxic-Ischemic Brain Injury in Neonatal Rats

Nitric oxide and excitatory amino acids contribute to hypoxic-ischemic brain injury. Agmatine, an endogenous neurotransmitter or neuromodulator, is an inhibitor of nitric oxide synthase and an antagonist of N-methyl-d-aspartate receptors. Does agmatine reduce brain injury in the rat pup hypoxic-ischemic model? Seven-day old rat pups had right carotid arteries ligated followed by 2.5 h of hypoxia (8% oxygen). Agmatine or vehicle was administered by i.p. injection at 5 min after reoxygenation and once daily thereafter for 3 d. Brain damage was evaluated by weight deficit of the right hemisphere at 22 d after hypoxia by a blinded observer. Agmatine treatments significantly reduced weight loss in the right hemisphere from −30.5 ± 3.6% in vehicle-treated pups (n = 22) to −15.6 ± 4.4% in the group treated with 50 mg/kg (n = 18, p < 0.05) and to −15.0 ± 3.7% in the group treated with 100 mg/kg (n = 18, p < 0.05), but the group treated with 150 mg/kg showed no reduction. Other pups received agmatine or vehicle at 5 min after reoxygenation, and brain biochemistry was assessed. Levels of endogenous brain agmatine rose 2- to 3-fold owing to hypoxic-ischemic (3 h), whereas pups treated with agmatine (100 mg/kg) showed 50-fold higher brain agmatine levels (3 h). Agmatine (100 mg/kg) blocked a hypoxia-induced increase in brain nitric oxide metabolites at 6 h (vehicle-treated, +60.2 ± 15.2%; agmatine-treated, +4.2 ± 8.4%;p < 0.05). Agmatine thus reduces brain injury in the neonatal rat hypoxic-ischemic model, probably by blunting the rise in nitric oxide metabolites normally seen after hypoxia.

Possible involvement of protein kinases in physical dependence on opioids: studies using protein kinase inhibitors, H-7 and H-8

Effects of a cAMP-dependent protein kinase and protein kinase C inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) and a cAMP- and cGMP-dependent protein kinase inhibitor, H-8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide), on the behavioral signs of naloxone (an opioid receptor antagonist)-precipitated withdrawal syndrome and effects of H-7 on the change of protein kinase C activity in the pons/medulla region induced by morphine (a mu-opioid receptor agonist) or butorphanol (a mu/delta/kappa mixed opioid receptor agonist) were investigated in this study. Rats were intracerebroventricularly (i.c.v.) infused with morphine (26 nmol/microliters/h) or butorphanol (26 nmol/microliters/h) through osmotic minipumps for 3 days. In some groups, either saline or drug-treated groups were concomitantly infused with H-7 (1 and 10 nmol/microliters/h) or H-8 (10 nmol/microliters/h). The expression of physical dependence produced by morphine or butorphanol, as evaluated by naloxone (5 mg/kg i.p.)-precipitated withdrawal signs, was reduced by concomitant infusion of H-7 or H-8. In the same condition, morphine and butorphanol chronic treatment enhanced (28.1% and 26.3% enhancement over the saline-treated group, respectively) cytosolic protein kinase C activity in the pons/medulla, but not in the membrane fraction. Furthermore, concomitant infusion of H-7 inhibited the enhancement of protein kinase C activity. These results indicate that various types of protein kinases may play an important role in the development and/or expression of physical dependence on opioids. Among them, the enhancement of cytosolic protein kinase C activity in the pons/medulla region seems to be one of the major underlying mechanisms in opioid physical dependence.

A protein kinase inhibitor, H-7, inhibits the development of tolerance to opioid antinociception

To investigate the effect of a protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), on the development of tolerance to antinociception induced by morphine (mu-opioid receptor agonist) and butorphanol (mu/delta/kappa-mixed opioid receptor agonist), rats were infused i.c.v. with morphine, butorphanol and H-7 through osmotic minipumps for 3 days. Concomitant infusion of H-7 dose dependently inhibited the development of tolerance to i.c.v. morphine- and butorphanol-induced antinociception. These results suggest that protein kinases may play an important role in the development of opioid tolerance.

Agmatine reduces extracellular glutamate during pentylenetetrazole-induced seizures in rat brain: A potential mechanism for the anticonvulsive effects

Glutamate has been implicated in the initiation and spread of seizure activity. Agmatine, an endogenous neuromodulator, is an antagonist of NMDA receptors and has anticonvulsive effects. Whether agmatine regulate glutamate release, as measured by in vivo microdialysis, is not known. In this study, we used pentylenetetrazole (PTZ)-induced seizure model to determine the effect of agmatine on extracellular glutamate in rat brain. We also determined the time course and the amount of agmatine that reached brain after peripheral injection. After i.p. injection of agmatine (50 mg/kg), increase of agmatine in rat cortex and hippocampus was observed in 15 min with levels returning to baseline in one hour. Rats, naïve and implanted with microdialysis cannula into the cortex, were administered PTZ (60 mg/kg, i.p.) with prior injection of agmatine (100 mg/kg, i.p.) or saline. Seizure grades were recorded and microdialysis samples were collected every 15 min for 75 min. Agmatine pre-treatment significantly reduced the seizure grade and increased the onset time. The levels of extracellular glutamate in frontal cortex rose two- to three-fold after PTZ injection and agmatine significantly inhibited this increase. In conclusion, the present data suggest that the anticonvulsant activity of agmatine, in part, could be related to the inhibition glutamate release.

Grape seed extract suppresses lipid peroxidation and reduces hypoxic ischemic brain injury in neonatal rats.

Oxygen radicals play a crucial role in brain injury. Grape seed extract is a potent anti-oxidant. Does grape seed extract reduce brain injury in the rat pup? Seven-day-old rat pups had the right carotid arteries permanently ligated followed by 2.5 h of hypoxia (8% oxygen). Grape seed extract, 50 mg/kg, or vehicle was administered by i.p. 5 min prior to hypoxia and 4 h after reoxygenation and twice daily for 1 day. Brain damage was evaluated by weight deficit of the right hemisphere at 22 days following hypoxia and by histopathology. Grape seed extract reduced brain weight loss from 20.0+/-4.4% S.E.M. in vehicle pups (n=21) to 3.1+/-1.6% in treated pups (n=20, P<0.01). Grape seed extract improved the histopathologic brain score in cortex, hippocampus and thalamus (P<0.05 versus vehicle). Concentrations of brain 8-isoprostaglandin F2alpha and thiobarbituric acid reacting substances significantly increased due to hypoxic ischemia. Grape seed extract reduced this increase. Treatment with grape seed extract suppresses lipid peroxidation and reduces hypoxic ischemic brain injury in neonatal rat.

Neuroprotective Effects of Vascular Endothelial Growth Factor Following Hypoxic Ischemic Brain Injury in Neonatal Rats

Vascular Endothelial Growth Factor (VEGF) protects the brain against ischemic injury in adult animals. We evaluated whether VEGF has neuroprotective effects against hypoxic-ischemic (HI) brain injury in newborn rats. Seven-day-old rat pups had the right carotid artery permanently ligated followed by 140 min of hypoxia (8% oxygen). VEGF (5, 10, 20, or 40 ng) or vehicle was administered intracerebroventricularly 5 min after reoxygenation following HI. Brain damage was evaluated by weight loss of the right hemisphere at 22 d after HI and by gross and microscopic morphology. Body weight, rectal temperature, and mortality were not significantly different in the VEGF and vehicle treated groups. VEGF treatment increased brain VEGF levels at 15 min after injection. VEGF (10 and 20 ng) significantly reduced brain weight loss (p < 0.05) and gross brain injury (p < 0.05); however, treatment with 5 or 40 ng did not. VEGF (10 ng) also decreased brain damage assessed by histologic scoring. VEGF increased phosphorylation of protein kinase B (Akt) and extracellular-signal regulated kinase 1/2 (ERK1/2) in the cortex (p < 0.05). These results suggest that VEGF has neuroprotective effects in the neonatal rat HI model that may be related to activation of the Akt/ERK signaling pathway.

Nicotinamide reduces hypoxic ischemic brain injury in the newborn rat

Nicotinamide reduces ischemic brain injury in adult rats. Can similar brain protection be seen in newborn animals? Seven-day-old rat pups had the right carotid artery permanently ligated followed by 2.5 h of 8% oxygen. Nicotinamide 250 or 500 mg/kg was administered i.p. 5 min after reoxygenation, with a second dose given at 6 h after the first. Brain damage was evaluated by weight deficit of the right hemisphere at 22 days following hypoxia. Nicotinamide 500 mg/kg reduced brain weight loss from 24.6 +/- 3.6% in vehicle pups (n = 28) to 11.9 +/- 2.6% in the treated pups (n = 29, P < 0.01), but treatment with 250 mg/kg did not affect brain weight. Nicotinamide 500 mg/kg also improved behavior in rotarod performance. Levels of 8-isoprostaglandin F2alpha measured in the cortex by enzyme immune assay 16 h after reoxygenation was 115 +/- 7 pg/g in the shams (n = 6), 175 +/- 17 pg/g in the 500 mg/kg nicotinamide treated (n = 7), and 320 +/- 79 pg/g in the vehicle treated pups (n = 7, P < 0.05 versus sham, P < 0.05 versus nicotinamide). Nicotinamide reduced the increase in caspase-3 activity caused by hypoxic ischemia (P < 0.01). Nicotinamide reduces brain injury in the neonatal rat, possibly by reducing oxidative stress and caspase-3 activity.

Dexamethasone pre-treatment protects brain against hypoxic-ischemic injury partially through up-regulation of vascular endothelial growth factor A in neonatal rats.

Dexamethasone (Dex) provides neuroprotection against subsequent hypoxia ischemia (HI) in newborn rats, but the mechanism of this neuroprotection is not well understood. It is known that vascular endothelial growth factor A (VEGF) has neuroprotective effects. The objective of this study was to evaluate the role of the VEGF signaling pathway in the Dex-induced neuroprotection in newborn rats. Seven-day-old rat pups had the right carotid artery permanently ligated followed by 140 or 160 min of hypoxia (8% oxygen). Rat pups received two i.p. injections of either saline or Dex (0.25 mg/kg) at 24 and 4 h before HI exposure. To quantify the effects of a glucocorticoid receptor (GR) blocker, on postnatal day (PD) 6 and 15 min prior to Dex treatment rat pups received s.c. vehicle or RU486 (GR blocker, 60 mg/kg). After 24 h at PD 7, all rat pups had HI as described earlier. To quantify the effects of a VEGFR 2 blocker, at 24 h after Dex/Veh treatment (PD7), SU5416, a VEGFR 2 inhibitor or vehicle was injected intracerebroventricularly in the right hemisphere at 30 min before and 2 h after HI. Dex pre-treatment reduced brain injury and enhanced the HI-induced brain VEGF protein while a GR blocker inhibited these effects. Treatment with VEGFR 2 blocker decreased Dex-induced neuroprotection also. Dex pre-treatment enhanced the HI-induced increase in mRNA expression of VEGF splice variants and decreased the HI-induced reduction of Akt phosphorylation. Additionally, it also decreased HI-induced increase of caspase-3 activity and DNA fragments in neonatal rat brain. We conclude that Dex provides robust neuroprotection against subsequent HI in newborn rats via GR likely with the partial involvement of VEGF signaling pathway.

Repeated immobilization stress alters rat hippocampal and prefrontal cortical morphology in parallel with endogenous agmatine and arginine decarboxylase levels

Agmatine, an endogenous amine derived from decarboxylation of L-arginine catalyzed by arginine decarboxylase, has been proposed as a neurotransmitter or neuromodulator in the brain. In the present study, we examined whether agmatine has neuroprotective effects against repeated immobilization-induced morphological changes in brain tissues and possible effects of immobilization stress on endogenous agmatine levels and arginine decarboxylase expression in rat brains. Sprague-Dawley rats were subjected to 2h immobilization stress daily for 7 days. This paradigm significantly increased plasma corticosterone levels, and the glutamate efflux in the hippocampus as measured by in vivo microdialysis. Immunohistochemical staining with beta-tubulin III showed that repeated immobilization caused marked morphological alterations in the hippocampus and medial prefrontal cortex that were prevented by simultaneous treatment with agmatine (50mg/kg/day), i.p.). Likewise, endogenous agmatine levels measured by high-performance liquid chromatography in the prefrontal cortex, hippocampus, striatum and hypothalamus were significantly increased by immobilization, as compared to controls. The increased endogenous agmatine levels, ranging from 92 to 265% of controls, were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. These results demonstrate that the administration of exogenous agmatine protects the hippocampus and medial prefrontal cortex against neuronal insults caused by repeated immobilization. The parallel increase in endogenous brain agmatine and arginine decarboxylase protein levels triggered by repeated immobilization indicates that the endogenous agmatine system may play an important role in adaptation to stress as a potential neuronal self-protection mechanism.