Intrauterine growth restriction and neonatal hypoxic ischemic brain injury causes sex-specific long-term neurobehavioral abnormalities in rats.

There is a lack of knowledge of factors preventing an adequate response to moderate hypothermia after hypoxic ischemic (HI) brain injury. We hypothesized that growth restriction from reduced intrauterine perfusion would predispose neonatal rats to have a worse outcome with HI brain injury. IUGR was induced by placental insufficiency in dams at 14 days of gestation. HI was induced at postnatal day (P) 10 by permanent right carotid artery ligation followed by 90 min of hypoxia (8% oxygen). Tests for early brain injury and neurobehavioral outcomes were subsequently done. All statistical analysis was done using Two-way ANOVA; post hoc Holm-Sidak test. HI in control and IUGR groups decreased the success rate of the contralateral vibrissa-elicited forelimb test, increased response latency in movement initiation test and increased the time to finish elevated beam walk test at P40 and P60. IUGR augmented HI-induced abnormality in vibrissa-elicited forelimb test at P40 but showed higher success rate when compared to HI only group at P60. IUGR's negative effect on HI-induced changes on the elevated beam walk test was sex-specific and exaggerated in P60 males. Increased TUNEL positive cells in the cortex were noted at 72 h after in HI in control but not in IUGR groups. In conclusion, the consequences of IUGR on subsequent neonatal HI varied based on age, sex and outcomes examined, and overall, male sex and IUGR had worse effects on the long-term neurobehavioral outcomes following HI.

Corticosteroid responses following hypoxic preconditioning provide neuroprotection against subsequent hypoxic-ischemic brain injury in the newborn rats.

Limited research has evaluated the corticosteroids (CS) response in hypoxic preconditioning (PC) induced neuroprotection against subsequent hypoxic-ischemic (HI) brain injury in newborns. To measure, CS response to hypoxic PC, at postnatal day 6 (P6), rat pups were randomly divided into sham, NoPC (exposure to 21% O2) and PC (exposure to 8% O2 for 3h) groups. In a separate experiment, at P6, rat pups were randomly divided into three groups (sham, NoPC+HI, PC+HI). Rat pups in NoPC+HI and PC+HI groups, respectively had normoxic or hypoxic exposure for 3h at P6 and then had the right carotid artery permanently ligated followed by 140 min of hypoxia at P7 (HI). Plasma CS levels were measured at 0.5, 1, 3, 6 and 12h after hypoxic PC and hypoxic PC followed by HI. To investigate whether CS response to hypoxic PC provides neuroprotection against HI, at P6, rat pups were randomly divided into five groups. Fifteen minutes prior to PC or normoxic exposure, rat pups in DMSO+PC+HI and DMSO+NoPC+HI groups received DMSO while in RU486+PC+HI and RU486+NoPC+HI groups received RU486 (glucocorticoid receptor blocker, 60 mg/kg) s.c., respectively. Afterwards, rat pups were exposed to normoxia (DMSO+NoPC+HI, RU486+NoPC+HI) or hypoxia (DMSO+PC+HI, RU486+PC+HI) for 3h and then HI 24h later (P7). Rat pups at the corresponding age without any exposure to PC or HI or RU486/DMSO were used as sham. We found that hypoxic PC caused CS surge as well as augmented CS surge and preserved the glucocorticoid feedback regulation after HI. Hypoxic PC reduced HI induced early and delayed brain damage. RU486 partially but significantly inhibited hypoxic PC induced neuroprotection.

Dexamethasone but not the equivalent doses of hydrocortisone induces neurotoxicity in neonatal rat brain.

BACKGROUND: The use of dexamethasone (Dex) in premature infants to treat or prevent chronic lung disease adversely affects neurodevelopment. Recent clinical studies suggest that hydrocortisone (HC) is a safer alternative to Dex. We compared the effects of Dex and HC on neurotoxicity in newborn rats. METHODS: Rat pups of a neurodevelopmental stage equivalent to premature human neonates were administered Dex or HC either as a single dose on postnatal day (PD) 6, repeated doses on PD 4 to 6 or tapering doses at PD 3 to 6 by i.p. injection. Brain weight, caspase-3 activity, and apoptotic cells were measured at PD 7; learning capability, memory, and motor function were measured at juvenile age. RESULTS: Dex decreased both body and brain weight gain, while HC did not. Tapering and repeated doses of Dex increased caspase-3 activity, cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells but HC, except at high doses, did not. Dex impaired learning and memory capability at juvenile age, while the rats exposed to HC showed normal cognitive behavior. CONCLUSION: HC is probably safer to use than Dex in the immediate postnatal period in neonatal rats. Cautious extrapolation of these findings to human premature infants is required.

Dexamethasone-induced neuroprotection in hypoxic-ischemic brain injury in newborn rats is partly mediated via Akt activation.

Prior treatment with dexamethasone (Dex) provides neuroprotection against hypoxia ischemia (HI) in newborn rats. Recent studies have shown that the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway plays an important role in the neuroprotection. The objective of this study is to evaluate the role of the PI3K/Akt pathway in the Dex-induced neuroprotection against subsequent HI brain injury. Seven-day-old rat pups had the right carotid artery permanently ligated followed by 160min of hypoxia (8% oxygen). Rat pups received i.p. injection of either saline or Dex (0.25mg/kg) at 24 and 4h before HI exposure. To quantify the effects of a PI3K/Akt inhibitor, wortmannin (1µl of 1µg/µl) or vehicle was injected intracerebroventricularly in the right hemisphere on postnatal day 6 at 30min prior to the first dose of Dex or saline treatment. Dex pretreatment significantly reduced the brain injury following HI which was quantified by the decrease in cleaved caspase-3 protein as well as cleaved caspase-3 and TUNEL positive cells at 24h and percent loss of ipsilateral hemisphere weight at 22d after HI, while wortmannin partially reversed these effects. We conclude that Dex provides robust neuroprotection against subsequent HI in newborn rats in part via activation of PI3/Akt pathway.

Dexamethasone induces apoptosis of progenitor cells in the subventricular zone and dentate gyrus of developing rat brain.

The use of dexamethasone in premature infants to prevent and/or treat bronchopulmonary dysplasia adversely affects neurocognitive development and is associated with cerebral palsy. The underlying mechanisms of these effects are multifactorial and likely include apoptosis. The objective of this study was to confirm whether dexamethasone causes apoptosis in different regions of the developing rat brain. On postnatal day 2, pups in each litter were randomly divided into the dexamethasone-treated (n = 91) or vehicle-treated (n = 92) groups. Rat pups in the dexamethasone group received tapering doses of dexamethasone on postnatal days 3-6 (0.5, 0.25, 0.125, and 0.06 mg/kg/day, respectively). Dexamethasone treatment significantly decreased the gain of body and brain weight and increased brain caspase-3 activity, DNA fragments, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and cleaved caspse-3-positive cells at 24 hr after treatment. Dexamethasone increased cleaved caspse-3-positive cells in the cortex, thalamus, hippocampus, cerebellum, dentate gyrus, and subventricular zone. Double-immunofluorescence studies show that progenitor cells in the subventricular zone and dentate gyrus preferentially undergo apoptosis following dexamethasone exposure. These results indicate that dexamethasone-induced apoptosis in immature cells in developing brain is one of the mechanisms of its neurodegenerative effects in newborn rats.

Dexamethasone decreases insulin-like growth factor-I and -II via a glucocorticoid receptor dependent mechanism in developing rat brain.

OBJECTIVES: Dexamethasone (Dex) causes neurodegeneration in developing brain. Insulin-like growth factor-I (IGF-I) and -II (IGF-II) are potent neurotrophic and differentiation factors and play key roles in the regulation of growth and development of CNS. Current project evaluated the effects of Dex on IGF-I and -II in developing rat brains. MATERIAL AND METHODS: Sprague-Dawley rat pups in each litter were divided into vehicle (n=230) or Dex-treated (n=234) groups. Rat pups in the Dex group received one of the 3 different regimens of i.p. Dex: tapering doses (DexTD) on postnatal days (PD) 3 to PD 6 or repeated doses on PD 4 to PD 6 or single dose on PD 6. To quantify the glucocorticoid receptor (GR) blockade effect, rat pups in the DexTD group on PD 3 and 5 received vehicle or RU486 (GR blocker, 60 mg/kg) s.c., twenty minutes prior to Dex treatment. RESULTS: Dex decreased the gain of body and brain weight while RU486 inhibited these effects. RU486 also prevented the DexTD-induced increase in caspase-3 activity and reduction in IGF-I and -II proteins. Compared to the vehicle, the expression of mRNA of IGF-I and -II decreased at 24 h after DexTD treatment, while RU486 prevented this decrease on IGF-II but not IGF-I. CONCLUSIONS: Our findings indicate that Dex via GR decreases IGF-I and -II and causes neurodegeneration in the neonatal rat brain.

Hypoxic preconditioning provides neuroprotection and increases vascular endothelial growth factor A, preserves the phosphorylation of Akt-Ser-473 and diminishes the increase in caspase-3 activity in neonatal rat hypoxic-ischemic model.

Vascular endothelial growth factor A (VEGF) likely plays a role in the hypoxic preconditioning (PC) induced tolerance to subsequent hypoxic-ischemic (HI) injury to the brain. However, limited data is available concerning VEGF in the developing brain after HI following PC. Neuroprotection by VEGF involves activation of Akt which inhibits apoptotic processes that contribute significantly to the brain injury in neonatal HI. We evaluated whether PC provides neuroprotection and affects VEGF, Akt and caspase-3 following HI in the developing rat brain. Newborn rats (6 days) were subjected to normoxia (21% O(2)) or PC (8% O(2)) for 3h followed by 24h of reoxygenation. The rats then had the right carotid artery permanently ligated followed by 140 min of hypoxia (8% O(2)) (HI or PC+HI). Brains from rats at the corresponding age without any exposure to PC or HI were examined for comparison (Sham). PC significantly reduced brain damage as measured by weight loss of the right hemisphere at 22 days after HI and by gross and microscopic morphology. PC amplified and prolonged the induction of mRNA of VEGF splice variants measured by real-time RT-PCR and enhanced the increase in VEGF protein measured by ELISA in brain following HI. PC preserved the phosphorylation of Akt-Ser-473 and diminished the increase in caspase-3 activity in brain following HI. We conclude that PC provides neuroprotection and augments and preserves the increase in VEGF following HI in the newborn rat brain which may play an important role in neuroprotection.

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.

D-Amino acids in rat brain measured by liquid chromatography/tandem mass spectrometry.

Previous work has established that D-amino acids including D-serine (D-Ser) and D-aspartic acid (D-Asp) fulfill specific biological functions in the brain. In this work, the levels and anatomical distribution of d-amino acids in rat brain were determined by using an advantageous liquid chromatography/tandem mass spectrometric analytical method. The study was focused on D-Ser, D-Asp, and D-glutamic acid (D-Glu) because of the significance of L-Asp, L-Glu, and D-Ser in the nervous system. Prenatal, postnatal pups, and 90-day old rats were studied. Results indicated that D-Asp and D-Ser occurred in rat brain at the microg/g tissue level. However, D-Glu was not detected (< 110 ng/g tissue). Throughout the developmental stages d-Asp content in rat brain decreased rapidly from 9.42% of total Asp in 5-day prenatal rats to an undetectable level (< 150 ng/g tissue) in 90-day old rats. In contrast, D-Ser level increased gradually throughout the developmental stages. D-Ser percentage (D-Ser/(D-Ser + L-Ser)) changed from 4.94% in 5-day prenatal rats to 13.7% in 90-day old rats. Regional levels of D-Ser were found to be significantly higher in cortex, striatum, and hippocampus than in thalamus. D-Ser was not detected in cerebellum (< 172ng/g tissue).

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.

Neuroprotective effects of sodium orthovanadate after hypoxic-ischemic brain injury in neonatal rats.

Sodium orthovanadate (SOV), a competitive inhibitor of protein tyrosine phosphatases, is neuroprotective in adult animals following an ischemic event. The present study evaluated whether SOV might be protective in a rat pup hypoxic-ischemic (HI) model. Seven-day-old rat pups had the right carotid artery permanently ligated followed by 140 min of hypoxia (8% oxygen). SOV 1.15, 2.3, 4.6, 9.2 or 18.4 mg/kg and vehicle were administered by i.p. injection at 5 min after reoxygenation. Brain damage was evaluated by weight loss of the right hemisphere at 22 days after hypoxia and by gross and microscopic morphology. SOV lowered blood glucose at doses of 1.15, 2.3 and 4.6 mg/kg and induced toxic effects at 9.2mg/kg. The doses of 2.3 and 4.6 mg/kg of SOV significantly reduced brain weight loss (p<0.05), but treatment with 1.15 or 9.2mg/kg did not. SOV 4.6 mg/kg also improved the histopathologic score and diminished the HI induced reduction of Akt and ERK-1/2 phosphorylation in the cortex (p<0.05) and increased the density of BrdU-positive cells in the subventricular zone (p<0.01). In conclusion, SOV has neuroprotective effects in the neonatal rat HI model partially mediated by activating Akt and ERK-1/2 pathways.

Grape seed extract given three hours after injury suppresses lipid peroxidation and reduces hypoxic-ischemic brain injury in neonatal rats.

We have reported that pretreatment with grape seed extract (GSE), a potent antioxidant, is neuroprotective. This study examined whether treatment after injury with GSE is protective. Seven-day-old rat pups had the right carotid artery ligated, and then 2.5 h of 8% oxygen. GSE (50 mg/kg) or vehicle was administered by i.p. initial injection at 5 min to 5 h after reoxygenation, with an additional three doses within 26 h after injury. Brain damage was evaluated by weight deficit of the right hemisphere at 22 d after hypoxia. Treatment at 3 h after reoxygenation reduced brain weight loss from 21.0 +/- 3.3% in vehicle-treated pups (n = 31) to 11.4 +/- 2.8% in treated pups (n = 31, p < 0.05). GSE lowered body temperature, but reduced brain injury even when body temperature was controlled. GSE reduced neurofunctional abnormalities caused by the hypoxia-ischemia (HI). GSE reduced a HI induced increase in 8-isoprostaglandin F2alpha (8-isoPGF2alpha) and reduced an HI-induced increase in the proapoptotic protein c-jun in the brain cortex. GSE up to 3 h after reoxygenation reduces brain injury in rat pups, probably by suppressing lipid peroxidation and the proapoptotic protein c-jun.

Assay of trace D-amino acids in neural tissue samples by capillary liquid chromatography/tandem mass spectrometry.

A sensitive chiral capillary HPLC-MS/MS method well suited for the determination of amino acid enantiomers in biological samples was developed. The method involved precolumn derivatization of the sample with 7-fluoro-4-nitrobenzoxadiazole (NBD-F). After derivatization, NBD-amino acids were stacked on a C18 reversed-phase extraction microcolumn, thus enriching and cleaning up the analytes. Various chiral stationary phases (CSPs) including cyclodextrin-bonded silica, Pirkle-type, vancomycin, and teicoplanin-bonded silica particles were evaluated for resolving NBD-F tagged amino acid enantiomers with mobile phases compatible with MS detection. It was found that only teicoplanin aglycon CSP provided sufficient resolution of NBD-Asp and NBD-Ser enantiomers to quantify trace levels of D-Asp and D-Ser in tissue samples. MS/MS detection of NBD-amino acid derivatives was very sensitive and selective. The high selectivity allowed the use of a stable isotope-labeled analyte analogue (i.e., L-aspartic acid-2,3,3-d3) as internal standard for the quantitation to improve assay reproducibility and reliability. Neural tissue samples dissected from rat brain and the central nervous system (CNS) of Aplysia californica, a widely used neuronal model, were analyzed to determine the chirality of glutamic acid (Glu), aspartic acid (Asp), and serine (Ser). The former two are major excitatory amino acids in the brain, and the last one has been recently identified as a neuromodulator. Both D-Ser and D-Asp were detected in rat brain. While the D-Asp level decreased rapidly through the developmental stages of the rat, the D-Ser level increased steadily from 82.3 microg/g of wet tissue in 3-day prenatal rats to 241.3 microg/g of wet tissue in 90-day-old rats. Interestingly, no D-Ser was detected in the CNS of Aplysia, a "primitive" invertebrate. However, the D-Asp level in this animal was found to be high. In a particular connective nerve sample, D-Asp was at 323.2 microg/g of wet tissue and constituted 60.2% of total Asp. D-Glu was not detected either in rat brain or in Aplysia's CNS.

N-tosyl-L-phenylalanyl-chloromethyl ketone reduces ceramide during hypoxic-ischemic brain injury in newborn rat.

N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK) suppresses apoptosis and protects neurons from damage in animal models. TPCK is thought to act by inhibiting ceramide production by sphingomyelinase. Ceramide is a proapoptotic intracellular signal that is involved in the cerebral ischemia. We wished to see whether ceramide contributes to TPCK's neuroprotective effects in vivo. Seven-day-old rat pups had the right carotid arteries permanently ligated followed by 2.5 h of hypoxia (8% oxygen). TPCK (10 mg/kg, n=62) or vehicle (n=63) was administered by i.p. 5 min prior to hypoxia. The level of ceramide in brain cortex both in lesioned and unlesioned hemispheres was measured at 8 h, 18 h, 24 h, 2 and 5 days after hypoxia-ischemia using reversed phase high performance liquid chromatography. The level of ceramide significantly increased due to hypoxic-ischemia at 18, 24 h and 2 days after hypoxia (P<0.05 or P<0.01) but not at 8 h or 5 days after hypoxia as compared to the contralateral hemisphere or a sham group. Pretreatment with TPCK reduced this increase. We also examined the level of sphingomyelin and the activities of the ceramide synthesizing sphingomyelinase enzymes by thin layer chromatography. The activities of acidic and neutral sphingomyelinase significantly increased due to hypoxic ischemia at 24 h after hypoxia. TPCK significantly reduced this increase (P<0.05 vs. vehicle) but did not affect the level of sphingomyelin. The results are consistent with the hypothesis that ceramide is involved in TPCK's neuroprotective effects in hypoxic-ischemic brain injury in the newborn rat.

Down-regulation of phospholipase D2 mRNA in neonatal rat brainstem and cerebellum after hypoxia-ischemia.

Phospholipase D (PLD) and phosphatidylcholine (PC) were implicated in apoptosis and cancer. However, direct evidence on the role of PLD in the cause of apoptosis remains obscure. It was recently reported that apoptosis and necrosis could be induced in the cerebellum and brainstem after focal cerebral hypoxic-ischemic (HI) injury. It was found that apoptosis could be enhanced by farnesol inhibition of PLD signal transduction. Whereas it was shown that highly invasive cancer cell line depends on PLD activity for survival when deprived of serum growth factors. Based on these reports, it is postulated that apoptosis in the cerebellum and brainstem induced after focal cerebral HI treatment may be caused by faulty PLD expression. This is consistent with a report that PLD1 activity and mRNA levels were down-regulated during apoptosis. To test this hypothesis, Northern blotting was used to examine PLD2 mRNA expression after focal cerebral HI. The results show that both PLD2 mRNA 10.8 and 3.9 kb transcripts were significantly decreased by as much as 37% in the brainstem and cerebellum areas 3 h after HI compared to the control, concur with previous report of decreasing PLD activity after ischemia. These PLD2 transcripts, however, were not significantly different from the control 3 days after HI, indicating that the decrease in PLD2 transcription after HI maybe a transient phenomenon. This is the first report to show that the loss of membrane integrity resulting from deprivation of energy and growth factors after HI could cause decrease in PLD2 transcription that promotes apoptosis. The hypothetic role of PLD2 and the mechanism leading to apoptosis remains to be further elucidated.

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.

1-Benzyl-1,2,3,4-tetrahydroisoquinoline passes through the blood-brain barrier of rat brain: an in vivo microdialysis study.

Previous work has established that 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BnTIQ) causes a parkinsonian syndrome in rats. The present study reports the blood-brain barrier (BBB) permeability of 1-BnTIQ in freely moving rats with the aid of in vivo microdialysis-based measurements. The microdialysis probe was implanted in the frontal cortex of rat brain. Brain dialysate samples were analyzed using an HPLC-MS/MS assay. 1-BnTIQ, when administered i.p., dose-dependently appeared in brain extracellular fluid (ECF), reaching a maximum concentration after about 40 min. Two other tetrahydroisoquinoline derivatives, 1,2,3,4-tetrahydroisoquinoline (TIQ) and 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline [salsolinol (SAL)], served as positive and negative controls, respectively. The results confirmed an earlier report that SAL does not reach the brain after i.p. administration. In contrast, TIQ readily passed through the BBB. The brain dialysate concentration of 1-BnTIQ was about 24% that of TIQ when administered i.p. at the same dose. Both of them decreased quickly with a half-life of about 50 min.

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.

Apoptosis and necrosis in developing cerebellum and brainstem induced after focal cerebral hypoxic-ischemic injury.

Focal cerebral hypoxia-ischemia due to isolated vascular insufficiency is well known to cause ipsilateral, but not contralateral, cerebral apoptosis. Hypoxic-ischemic damage to the cerebellum and brainstem in such a model has not been established. This experimental rodent study demonstrates, through deoxyribonucleic acid fragmentation and terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate-digoxigenin nick end labeling analysis, that neuronal cells in these infratentorial regions also suffer mild apoptosis and necrosis after focal cerebral hypoxic-ischemic injury in the newborn rat. These data provide additional insight into the mechanisms of neurological injury in the cerebellum and brainstem areas resulting from a focal cerebral hypoxic-ischemic insult and demonstrate that future therapeutic interventions for hypoxic-ischemic encephalopathy system should deal with the entire central nervous system.