FTY720 inhibits germ cell apoptosis in testicular torsion/detorsion.

BACKGROUND: Testicular torsion/detorsion (T/D) can induce germ cells apoptosis, which may lead to impairment of spermatogenesis. FTY720, an agonist of the sphingosine-1-phosphate receptor 1 (S1PR1), inhibits apoptosis in ischemic stroke. We examined whether FTY720 could mitigate germ cell apoptosis in testicular T/D rats. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were allocated to receive testicular T/D (the T/D group), T/D plus FTY720 (the T/D-FTY group), or T/D plus FTY720 plus the potent S1PR1 antagonist VPC23019 (the T/D-FTY-VPC group; n = 6 in each group). Sham control groups were run simultaneously. At 24 h after detorsion, rats were euthanized. RESULTS: Our data revealed that, in the ipsilateral twisted testes, sperm counts and expression of the S1PR1 of the T/D and the T/D-FTY-VPC groups were significantly lower than those of the T/D-FTY group (all P < 0.001). In contrast, signals of apoptotic cells stained by terminal deoxynucleotidyl transferase dUTP nick end labeling and the proapoptotic protein cleaved caspase-3 of the T/D, and the T/D-FTY-VPC groups were significantly stronger than those of the T/D-FTY group. Moreover, the terminal deoxynucleotidyl transferase dUTP nick end labeling signals mainly localized to germ cells. CONCLUSIONS: FTY720 could mitigate testicular T/D-induced germ cell apoptosis, and the mechanisms may involve the S1PR1.

Regulation of Intermittent Hypoxia on Brain Dopamine in Amphetaminized Rats.

We investigated intermittent hypoxia (IH) on dopamine (DA) release in rat brain treated with or without amphetamine (AMPH). Rats were divided into four groups including normoxia, IH, AMPH, and AMPH + IH treatments. The cerebrospinal fluid (CSF) was collected and the DA levels were detected by high performance liquid chromatography (HPLC). The plasma prolactin (PRL) concentration was measured by radioimmunoassay (RIA). We found that IH reduced basal DA concentration in media prefrontal cortex (mPFC), but increased that in striatum, where DA level was also increased in rats treated with AMPH or AMPH + IH. Angiotensin II (Ang II) increased the DA release in mPFC and striatum and this effect was enhanced in AMPH + IH group. The stimulatory effect of IH on plasma PRL was attenuated in presence of AMPH. Tyrosine hydroxylase (TH) expression was decreased by IH, but increased by AMPH + IH in mPFC. IH or AMPH treatment decreased the expression of vesicular monoamine transporter-2 (VMAT-2) in rat brain. These data suggested that IH altered the DA release and changed the protein expression levels in different parts of rat brain treated with AMPH. IH may play a role in regulating DA metabolism in AMPH addiction.

FTY720 mitigates torsion/detorsion-induced testicular injury in rats.

BACKGROUND: FTY720, a sphingosine-1-phosphate (S1P) receptor agonist, possesses potent anti-inflammation capacity. We evaluated the therapeutic potentials of FTY720 against testicular injury induced by testicular torsion and/or detorsion (T/D). MATERIALS AND METHODS: Young adult male Sprague-Dawley rats were allocated to receive T/D (the T/D group) and T/D plus FTY720 (4 mg/kg, the T/D-FTY group, n = 6 in each group). To investigate the possible roles of the S1P receptors, another group of rats received T/D plus FTY720 plus the potent S1P receptor antagonist VPC23019 (1 mg/kg, the T/D-FTY-VPC group, n = 6). FTY720 was administered immediately before testicular detorsion, and VPC23019 was administered 30 min before FTY720. Another set of rats that received sham operation, immediately followed by injection of normal saline, FTY720, or FTY720 plus VPC23019, served as control groups. Sham control groups were run simultaneously. After euthanization, levels of testicular injury were measured. RESULTS: Histologic findings revealed severe testicular injury changes in both the T/D and T/D-FTY-VPC groups and moderate testicular injury changes in the T/D-FTY group. In addition, malondialdehyde activity (oxidative status), concentration of interleukin-1ß (inflammation index), myeloperoxidase activity (neutrophil infiltration index), and wet-to-dry weight ratio (tissue edema index) of both the T/D and T/D-FTY-VPC groups were significantly higher than those of the T/D-FTY group. These data confirmed the protective effects of FTY720 against testicular T/D. Moreover, antagonizing the S1P receptors could reverse the protective effects of FTY720. CONCLUSIONS: FTY720 significantly mitigated testicular injury induced by testicular T/D. The mechanisms may involve activating the S1P receptors.

Exploration of approaches to adjusting brand-name drug prices in Mainland of China: based on comparison and analysis of some brand-name drug prices of Mainland and Taiwan, China.

BACKGROUND: Under the circumstance of the New Medical Reform in Mainland of China, lowering drug prices has become an approach to relieving increase of medical expenses, and lowering brand-name medication price is a key strategy. This study, by comparing and analyzing brand-name medication prices between Mainland of China and Taiwan, explores how to adjust brand-name medication prices in Mainland of China in the consideration of the drug administrative strategies in Taiwan. METHODS: By selecting brand-name drug with generic name and dose types matched in Mainland and Taiwan, calculate the average unit price and standard deviation and test it with the paired t-test. In the mean time, drug administrative strategies between Mainland and Taiwan are also compared systematically. RESULTS: Among the 70 brand-name medications with generic names and matched dose types, 54 are at higher prices in Mainland of China than Taiwan, which is statistically significant in t-test. Also, among the 47 medications with all of matched generic names, dose types, and manufacturing enterprises, 38 are at higher prices in Mainland than Taiwan, and the gap is also statistically significant in t-test. In Mainland of China, brand-name medication took cost-plus pricing and price-based price adjustment, while in Taiwan, brand-name medication took internal and external reference pricing and market-based price adjustment. CONCLUSIONS: Brand-name drug prices were higher in Mainland of China than in Taiwan. The adjustment strategies of drug prices are scientific in Taiwan and are worth reference by Mainland of China.

Integrated wireless fast-scan cyclic voltammetry recording and electrical stimulation for reward-predictive learning in awake, freely moving rats.

OBJECTIVE: Fast-scan cyclic voltammetry (FSCV) is commonly used to monitor phasic dopamine release, which is usually performed using tethered recording and for limited types of animal behavior. It is necessary to design a wireless dopamine sensing system for animal behavior experiments. APPROACH: This study integrates a wireless FSCV system for monitoring the dopamine signal in the ventral striatum with an electrical stimulator that induces biphasic current to excite dopaminergic neurons in awake freely moving rats. The measured dopamine signals are unidirectionally transmitted from the wireless FSCV module to the host unit. To reduce electrical artifacts, an optocoupler and a separate power are applied to isolate the FSCV system and electrical stimulator, which can be activated by an infrared controller. MAIN RESULTS: In the validation test, the wireless backpack system has similar performance in comparison with a conventional wired system and it does not significantly affect the locomotor activity of the rat. In the cocaine administration test, the maximum electrically elicited dopamine signals increased to around 230% of the initial value 20 min after the injection of 10 mg kg(-1) cocaine. In a classical conditioning test, the dopamine signal in response to a cue increased to around 60 nM over 50 successive trials while the electrically evoked dopamine concentration decreased from about 90 to 50 nM in the maintenance phase. In contrast, the cue-evoked dopamine concentration progressively decreased and the electrically evoked dopamine was eliminated during the extinction phase. In the histological evaluation, there was little damage to brain tissue after five months chronic implantation of the stimulating electrode. SIGNIFICANCE: We have developed an integrated wireless voltammetry system for measuring dopamine concentration and providing electrical stimulation. The developed wireless FSCV system is proven to be a useful experimental tool for the continuous monitoring of dopamine levels during animal learning behavior studies of freely moving rats.

Baicalein protects against retinal ischemia by antioxidation, antiapoptosis, downregulation of HIF-1α, VEGF, and MMP-9 and upregulation of HO-1.

PURPOSE: Retinal ischemia-associated ocular disorders are vision threatening. This study examined whether the flavonoid baicalein is able to protect against retinal ischemia/reperfusion. METHODS: Using rats, the intraocular pressure was raised to 120 mmHg for 60 min to induce retinal ischemia. In vitro, an ischemic-like insult, namely oxidative stress, was established by incubating dissociated retinal cells with 100 µM ascorbate and 5 µM FeSO4 (iron) for 1 h. The rats or the dissociated cells had been pretreated with baicalein (in vivo: 0.05 or 0.5 nmol; in vitro: 100 µM), vehicle (1% ethanol), or trolox (in vivo: 5 nmol; in vitro: 100 µM or 1 mM). The effects of these treatments on the retina or the retinal cells were evaluated by electrophysiology, immunohistochemistry, terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) staining, Western blotting, or in vitro dichlorofluorescein assay. In addition, real-time-polymerase chain reaction was used to assess the retinal expression of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), vascular endothelium growth factor (VEGF), and heme oxygenase-1 (HO-1). RESULTS: The retinal changes after ischemia included a decrease in the electroretinogram b-wave amplitude, a loss of choline acetyltransferase immunolabeling amacrine cell bodies/neuronal processes, an increase in vimentin immunoreactivity, which is a marker for Müller cells, an increase in apoptotic cells in the retinal ganglion cell layer linked to a decrease in the Bcl-2 protein, and changes in the mRNA levels of HIF-1α, VEGF, MMP-9, and HO-1. Of clinical importance, the ischemic detrimental effects were concentration dependently and/or significantly (0.05 nmol and/or 0.5 nmol) altered when baicalein was applied 15 min before retinal ischemia. Most of all, 0.5 nmol baicalein significantly reduced the upregulation of MMP-9; in contrast, 5 nmol trolox only had a weak attenuating effect. In dissociated retinal cells subjected to ascorbate/iron, there was an increase in the levels of reactive oxygen species, which had been significantly attenuated by 100 µM baicalein and trolox (100 µM or 1 mM; a stronger antioxidative effect at 1 mM). CONCLUSIONS: Baicalein would seem to protect against retinal ischemia via antioxidation, antiapoptosis, upregulation of HO-1, and downregulation of HIF-1α, VEGF, and MMP-9. The antioxidative effect of baicalein would appear to play a minor role in downregulation of MMP-9.

Resveratrol mitigates rat retinal ischemic injury: the roles of matrix metalloproteinase-9, inducible nitric oxide, and heme oxygenase-1.

PURPOSE: Retinal ischemia-associated ocular disorders, such as retinal occlusive disorders, neovascular age-related macular degeneration, proliferative diabetic retinopathy, and glaucoma are vision-threatening. In this study, we examined whether and by what mechanisms resveratrol, a polyphenol found in red wine, is able to protect against retinal ischemia/reperfusion injury. METHODS: In vivo rat retinal ischemia was induced by high intraocular pressure (HIOP), namely, 120 mmHg for 60 min. The mechanism and management was evaluated by electroretinogram (ERG) b-wave amplitudes measurement, immunohistochemistry, and real-time polymerase chain reaction. RESULTS: The HIOP-induced retinal ischemic changes were characterized by a decrease in ERG b-wave amplitudes, a loss of choline acetyltransferase immunolabeling of amacrine cell bodies/neuronal processes, and increased vimentin immunoreactivity, which is a marker of Müller cells, together with upregulation of matrix metalloproteinase-9 (MMP-9), heme oxygenase-1 (HO-1), and inducible nitric oxide (iNOS), and downregulation of Thy-1, both at the mRNA level. The detrimental effects due to the ischemia were concentration-dependent (weaker effect at 0.05 nmole) and/or significantly (at 0.5 nmole) altered when resveratrol was applied 15 min before or after retina ischemia. CONCLUSION: This study supports the hypothesis that resveratrol may be able to protect the retina against ischemia by downregulation of MMP-9 and iNOS, and upregulation of HO-1.

The effects and underlying mechanisms of S-allyl l-cysteine treatment of the retina after ischemia/reperfusion.

PURPOSE: Retinal ischemia-associated ocular disorders are vision-threatening. The aim of the present study was to examine whether S-allyl l-cysteine (SAC) is able to protect against retina ischemia/reperfusion injury. METHODS: In vivo, retinal ischemia in the rat was induced by raising intraocular pressure (IOP) to 120 mmHg for 60 min. In vitro, an ischemic-like insult, namely oxidative stress, was established by incubating retinal ganglion cell-5 (RGC-5) with 500 µM H(2)O(2) for 24 h. The mechanisms involved in these processes were evaluated by electrophysiology, immunohistochemistry, and molecular biological approaches. RESULTS: The retinal changes caused by the high IOP were characterized by a decrease in electroretinogram b-wave amplitudes, a loss of choline acetyltransferase immunolabeling amacrine cell bodies/neuronal processes, and an upregulation of the mRNA levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelium growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9). The increased protein levels of HIF-1α, VEGF, and MMP-9 were also seen in RGC-5 cells subjected to defined oxidative stress. Of clinical importance, the ischemic/ischemic-like detrimental effects were concentration-dependently (least effect at 25 µM) and/or significantly (50 and/or 100 µM) blunted when SAC was applied 15 min before retinal ischemia or ischemic-like insult, respectively. CONCLUSION: SAC would seem to protect against retinal ischemia by acting as an antioxidant and inhibiting the upregulation of HIF-1α, VEGF, and MMP-9.

Baicalein significantly protects human retinal pigment epithelium cells against H2O2-induced oxidative stress by scavenging reactive oxygen species and downregulating the expression of matrix metalloproteinase-9 and vascular endothelial growth factor.

PURPOSE: Age-related macular degeneration is a leading cause of blindness in the elderly. At a later stage, neovascular or exudative age-related macular degeneration can lead to severe central vision loss that is related to aging-associated cumulative oxidative stress of the human retinal pigment epithelium (hRPE) cells. Early prevention with antioxidants is mandatory. The aim of this study was to determine whether and how baicalein can act as an antioxidant. METHODS: The methods used included lactate dehydrogenase, 2',7'-dichloro-fluorescein diacetate, or enzyme-linked immunosorbent assay to measure cell viability, oxygen free radical levels, or the levels of vascular endothelial growth factor (VEGF)/matrix metalloproteinase-9 (MMP-9), respectively. RESULTS: H2O2 dose-dependently reduced the cell viability of hRPE cells. This negative effect was dose-dependently (with a lower effect at 20µM) and significantly counteracted by pretreatment with baicalein (50µM). Treatment with H2O2 significantly stimulated the formation of oxygen free radicals. This increase was dose-dependently and significantly blunted by baicalein. Further, treatment with a sublethal dose of H2O2 was associated with an upregulation in the levels of VEGF and MMP-9. The increases in these proteins were also dose-dependently (with a lower effect at 20µM) and significantly (50µM) blunted by pretreatment with baicalein. CONCLUSION: This study supports an antioxidative role for baicalein whereby it protects hRPE cells against H2O2-induced oxidative stress by downregulating the levels of VEGF and MMP-9, which are increased by H2O2.

Therapeutic effects and mechanisms of action of mannitol during H2O2-induced oxidative stress in human retinal pigment epithelium cells.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. At a later stage, neovascular or exudative AMD can lead to severe central vision loss that is related to aging-associated cumulative oxidative stress of the human retinal pigment epithelium (hRPE) and choroid capillary. Early prevention with antioxidants is mandatory. The aim of this study was to determine whether and how mannitol can act as an antioxidant. METHODS: The methods used included measurements of cell viability, oxygen free radical (OFR) levels, lipid peroxide (LP) levels, and OFR-related enzyme protein levels. RESULTS: H(2)O(2) dose-dependently reduced the cell viability of hRPE cells. This negative effect was significantly counteracted by pretreatment with mannitol (1 mM). H(2)O(2) significantly stimulated the formation of OFR and LP. These increases were dose-dependently and significantly blunted by mannitol. Furthermore, treatment with H(2)O(2) was associated with a reduction in the level of catalase, but not of manganese superoxide dismutase (MnSOD). In contrast, it was shown that mannitol protected hRPE cells against the H(2)O(2)-induced oxidative stress by increasing the level of catalase, but not the level of MnSOD. CONCLUSION: This study supports an antioxidative role for mannitol that acts through up-regulating the level of catalase, which is decreased by H(2)O(2).

Attenuation of estradiol on the reduction of striatal dopamine by amphetamine in ovariectomized rats.

Amphetamine (AMPH) is a highly addictive drug of abuse which exhibits toxicity to dopaminergic neurons in long-term abusers. Estrogen seems to show neuroprotection in dopamine (DA) deficit caused by AMPH. The present study was to investigate the effects of estradiol on the levels of striatal DA in ovariectomized (Ovx) rats treated with or without AMPH. Female rats were Ovx for 2 weeks before administration of AMPH (5 mg/kg/day, i.p.) with or without 17beta-estradiol benzoate (EB) (25 microg/kg/day, s.c.) for 7 days. The striatal tissues were collected, homogenized with DA mobile phase, and centrifuged. The concentrations of DA in the supernatants were detected by HPLC. The protein expressions of dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT-2), and tyrosine hydroxylase (TH) were analyzed by Western blotting. The results indicated that AMPH could attenuate DA level significantly in striatum (P < 0.01). Comparing to control groups, administration of either EB or EB plus AMPH increased DA level (P < 0.01). The protein expression of striatal DAT was significant greater (P < 0.01) in rats treated with AMPH plus EB than AMPH treated animals. These results suggest that the DA levels in striatum can be enhanced by EB via an increase of DAT expression following administration of AMPH.

Ferulic acid, but not tetramethylpyrazine, significantly attenuates retinal ischemia/reperfusion-induced alterations by acting as a hydroxyl radical scavenger.

PURPOSE: Ischemia plays an important role in glaucomatous optic neuropathy and retinal vascular occlusive disorders, which renders investigation vital. METHODS: Retinal ischemia was induced by raising intraocular pressure to 120 mmHg. Its mechanism and management was evaluated by measuring (*)OH levels, electroretinogram (ERG) b-wave amplitudes, immunohisto-chemistry, and reverse transcriptase polymerase chain reaction. RESULTS: Ischemia for 45, 60, and 75 min caused significant and time-dependent increased (*)OH levels, which might contribute to retinal ischemic injures. Specifically, 60 min of ischemia plus reperfusion, causing moderate oxidative stress, resulted in retinal changes that were characterized by decreased ERG b-wave amplitudes, loss of choline acetyltransferase immunolabeled amacrine cell bodies/neuronal processes, downregulated Thy-1 m-RNA levels (indexing retinal ganglion cells; RGCs), and reduced thickness of the Thy-1 immunolabeled RGC and inner plexiform layers. Of clinical importance, this is the first study to show that ischemic detrimental effects are significantly blunted when 0.5 nmol of ferulic acid, one active ingredient of Ligusticum walliichi (Chuanxiong), was applied 24 h before retinal ischemia. Further, but not to a significant level, 0.5 nmole of tetramethylpyrazine, another Chuanxiong-active component, showed such an ameliorating trend. Moreover, the 60-min ischemia-induced significant increase in (*)OH production was significantly attenuated by FA. CONCLUSIONS: FA is able to protect against retinal ischemia and possibly glaucoma by, at least in part, acting as a (*)OH scavenger.

Difference in magnitude of psychostimulant-induced extracellular norepinephrine in the ventral tegmental area contributes to discrepant prefrontal dopamine outflow.

The dopamine (DA) efflux in the medial prefrontal cortex (mPFC) can be modulated by the interaction between afferent norepinephrine (NE) and somatodendritic DA in the ventral tegmental area (VTA). However, it is unclear how locally administered amphetamine (AMPH) or cocaine in the VTA results in discrepant response of DA efflux in the mPFC. In this study, intra-VTA infusion of AMPH (1000 microM) or cocaine (200 microM) in anesthetized rats was employed to study the concurrent profile of extracellular DA level in the VTA and mPFC. In addition, the extracellular NE levels during the intra-VTA infusion of these two psychostimulants were analyzed to compare their effects on prefrontal DA efflux. During the intra-VTA infusion of AMPH, both extracellular DA and NE increased significantly in the VTA (270 +/- 12% and 819 +/- 40%, respectively). Meanwhile, the DA efflux in the mPFC elevated significantly. During the intra-VTA infusion of cocaine, the extracellular DA and NE in the VTA also increased (271 +/- 21% and 150 +/- 15%, respectively). However, the DA efflux decreased significantly in the mPFC. Noteworthy, the increase of extracellular NE in the VTA was much more robust via AMPH infusion, as compared with cocaine. It is suggested that AMPH and cocaine enhance the extracellular NE concentrations in the VTA in different magnitudes, which in turn contribute to discrepant profiles of distal DA efflux in the mPFC.

Prefrontal dopamine efflux during exposure to drug-associated contextual cues in rats with prior repeated methamphetamine.

Conditioned stimulus-reward response and prefrontal dopamine efflux under context previously paired with methamphetamine administration were assessed in rats with or without prior sensitizing regimen. Sensitizing pretreatment was administered with methamphetamine (1mg/kg, every other day for six sessions) for behavioral sensitization. The animals received methamphetamine (1mg/kg) or saline injection (each for six sessions) to pair with distinct contexts on alternate days to induce conditioned place preference. Then, dopamine outflows in the medial prefrontal cortex were analyzed on the next day via microdialysis study as animals exposed to the methamphetamine or saline-paired context, respectively. Prefrontal DA efflux increased in those rats without sensitizing pretreatment, while they occupied the methamphetamine-paired chamber. The rats with prior sensitizing regimen demonstrated more robust conditioned place preference than those without pretreatment, however, their dopamine efflux was attenuated, while remaining in methamphetamine-paired context. It is suggested that the attenuated responsiveness of mesocortical dopamine transmission in prior sensitized rats may, at least in part, be responsible for their augmented conditioned place preference, which resulted from activation of related brain areas that together strengthen the associative learning to drug-related stimuli. This paradigm may reflect a dysregulated prefrontal function in the methamphetamine abusers.

A single intrategmental amphetamine exposure induces transient behavioral sensitization in the rats.

Repeated treatment with psychostimulant drugs induces enduring behavioral sensitization and neuroadaptations which may play an important role in the development of drug addiction. However, different number and time course in drug administration and various lengths of drug withdrawal were employed in the literature, and there were inconsistent findings in the profile of extracellular dopamine level related to behavioral sensitization. Therefore, the effects of the number of drug exposure and the length of drug withdrawal period on the sensitized behavioral response were investigated in this study. Various lengths of amphetamine (AMPH) withdrawal (1, 3 and 5 days) after a single local administration of AMPH to bilateral ventral tegmental area (VTA) were used to observe the locomotor activity response. Besides, different amounts of administration of intra-VTA AMPH were given (1, 2 and 3 times of injection) to monitor the profile of travel distance and stereotypic movements of rats after 7 days of drug withdrawal. An early and short-lived behavioral sensitization to the single intra-VTA AMPH administration was induced. In the repeated treatment group, more drug exposures were associated with escalating and robust levels of travel distance after 7 days of drug withdrawal. The authors speculated that the transient and, a later augmented locomotor activity response might represent respective phases in the development of behavioral sensitization, which in turn contributed to the formation of more lasting behavioral and neuroplastic changes associated with drug addiction.

Pulmonary transcription of CAT-2 and CAT-2B but not CAT-1 and CAT-2A were upregulated in hemorrhagic shock rats.

Hemorrhagic shock stimulates nitric oxide (NO) biosynthesis through upregulation of inducible NO synthase (iNOS) expression. Trans-membrane l-arginine transportation mediated by the isozymes of cationic amino acid transporters (e.g. CAT-1, CAT-2, CAT-2A, and CAT-2B) is one crucial regulatory mechanism that regulates iNOS activity. We sought to assess the effects of hemorrhage and resuscitation on the expression of these regulatory enzymes in hemorrhage-stimulated rat lungs. Twenty-four rats were randomized to a sham-instrumented group, a sustained shock group, a shock with blood resuscitation group, or a shock with normal saline resuscitation group. Hemorrhagic shock was induced by withdrawing blood to maintain MAP between 40 and 45mmHg for 60min. Resuscitation by infusing blood/saline mixtures (blood resuscitation group) or saline alone (saline resuscitation group) was then performed. At the end of the experiment (300min after hemorrhage began), rats were sacrificed and enzymes expression as well as pulmonary NO biosynthesis and lung injuries were assayed. Our data revealed that hemorrhage-induced pulmonary iNOS, CAT-2, and CAT-2B transcription which was associated with pulmonary NO overproduction and subsequent lung injury. Resuscitation significantly attenuated the hemorrhage-induced enzyme upregulation, pulmonary NO overproduction, and lung injury. Blood/saline mixtures were superior to saline as a resuscitation solution in treating hemorrhage-induced pulmonary NO overproduction and lung injury. Hemorrhage and/or resuscitation, however, did not affect the expression of pulmonary CAT-1 and CAT-2A. It is, therefore, concluded that the expression of pulmonary iNOS, CAT-2, and CAT-2B is inducible and that of CAT-1 and CAT-2A is constitutive in hemorrhagic shock rat lungs.

Determination of amphetamine in rat brain by in vivo microdialysis and ion-pairing liquid chromatography with electrospray tandem mass spectrometry.

An ion-pairing liquid chromatography/electrospray tandem mass spectrometry (LC/ES-MS/MS) method with in vivo microdialysis for the determination of amphetamine in rat brain has been developed. A microdialysis probe was surgically implanted into the striatum of the rat and artificial cerebrospinal fluid (aCSF) was used as the perfusion medium. Samples were collected and then analyzed off-line by LC/ES-MS/MS. A reversed-phase C18 column was employed for LC separation and MS/MS was utilized for detection. Trifluoroacetic acid (TFA) was added to the mobile phase (acetonitrile/water) as an ion-pairing reagent. Detection was by ES-MS/MS directly, and no post-column addition of organic modifier was needed. Dual linear ranges were determined from 0.1-0.5 microg/mL and 0.005-0.1 microg/mL, respectively. The detection limit, based on a signal-to-noise ratio of 3, was 0.001 microg/mL (5 nM). Good precision and accuracy were obtained. The applicability of this newly developed method was demonstrated by continuous monitoring of amphetamine concentrations in rat brain. Amphetamine reached a maximum concentration of 0.086 +/- 0.017 microg/mL over 20-40 min after a single 3.0 mg/kg intraperitoneal administration.

Neurochemical interaction between dopaminergic and noradrenergic neurons in the medial prefrontal cortex.

Growing evidence indicates that there is an interaction between the transmission of dopamine (DA) and norepinephrine (NE) in the noradrenergic and dopaminergic projections that converge in the medial prefrontal cortex (mPFC). The effects of the noradrenergic alpha1 and alpha2 receptors and the NE transporters on the DA outflow and those of the dopaminergic D1 and D2 receptors on NE release in the mPFC were investigated. Local infusions of NE (90, 150, and 300 nM) into the mPFC increased the extracellular release of DA in anesthetized rats. The alpha1 receptor antagonist (10 microM prazosin), but not the alpha2 receptor antagonist (100 microM piperoxan), blocked the NE-induced increase of DA in the mPFC. In addition, local infusion of alpha1 receptor agonist (10 microM phenylephrine) enhanced DA release in the mPFC. Local application of DA in different concentrations into the mPFC increased extracellular NE levels. Intra-mPFC infusion of a D1 receptor antagonist (10 nM SCH23390), inhibited the DA-induced increase of NE; this did not happen with a D2 receptor antagonist (1 nM eticlopride). Local administration of a selective NE uptake inhibitor (1 microM desmethylimipramine) into the mPFC increased the outflows of both DA and NE in the mPFC. However, co-infusion of DMI and prazosin blunted, but did not totally abolish, the DMI-increase in the extracellular levels of DA and NE. These results suggest that in the mPFC, 1) extracellular NE could enhance DA release by activating the alpha1 receptors; and 2) extracellular DA increased the extracellular levels of NE by activating the D1 receptors.

Determination of free-form amphetamine in rat brain by ion-pair liquid chromatography-electrospray mass spectrometry with in vivo microdialysis.

An ion-pair liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) method with in vivo microdialysis for the determination of free-form amphetamine in rat brain has been developed. A microdialysis probe was surgically implanted into the striatum of the rat and artificial cerebrospinal fluid (aCSF) was used as the perfusion medium. Samples were collected and then analyzed off-line by LC-ESI-MS. A reversed phase C18 column was employed for LC separation. Trifluoroacetic acid (TFA) was added in the mobile phase (acetonitrile-water, 10:90, v/v) as an ion-pair reagent. The ion-pair process disguises the protonated amphetamine cations from the ESI-MS electric field as neutral molecules. Post-column addition of volatile organic acid was utilized to minimize TFA signal suppression effect on ESI-MS detection. More than six-fold enhancement of ESI-MS response was achieved by the post-column addition of propionic acid. Good linearity (0.01-1.00 microg/ml, r2 = 0.99) and detection limit (0.002 microg/ml) were determined. Good precision and accuracy were obtained. The applicability of this newly developed method was demonstrated by continuous monitoring of amphetamine concentrations in rat brain after a single 3.0 mg/kg i.p. administration.

Simultaneous determination of free-form amphetamine in rat's blood and brain by in-vivo microdialysis and liquid chromatography with fluorescence detection.

The determination of amphetamine in rat's blood and brain simultaneously by micordiaysis technique and HPLC/fluorescence derivatization is described. Microdiaysis is used to sample rat's blood and brain fluid and the amphetamine content is evaluated by HPLC/fluorescence derivatization assay. Dansyl chloride is used as fluorescence derivatization reagent. The detection limit, linearity and precision associated with this assay were evaluated. Pharmarcokinetic parameters of amphetamine in rat's blood and brain are also reported.