Hameed Al-Sarraf

Polarized distribution of nucleoside transporters in rat brain endothelial and choroid plexus epithelial cells

This study investigated mRNA expression and protein localization of equilibrative and concentrative nucleoside transporters (ENTs, CNTs) in primary cultures of rat brain endothelial cells (RBEC) and rat choroid plexus epithelial cells (RCPEC). Reverse transcriptase PCR analysis revealed that RBEC and RCPEC contained mRNA for rENT1, rENT2 and rCNT2 and for rENT1, rENT2, rCNT2 and rCNT3, respectively. Immunoblotting of membrane fractions of RBEC, fresh RCPEC and primary cultures of RCPEC revealed the presence of rENT1, rENT2 and rCNT2 proteins in all samples. Measurement of [14C]adenosine uptake into cells grown as monolayers on permeable plastic supports revealed a polarized distribution of Na+‐dependent adenosine uptake in that CNT activity was associated exclusively in membranes of RBEC facing the lower chamber (which corresponds to the surface facing the interstitial fluid in situ) and in membranes of RCPEC facing the upper chamber (which corresponds to the surface facing the cerebrospinal fluid in situ). In both RBEC and RCPEC, adenosine uptake from the opposite chambers was Na+‐independent and partially inhibited by nitrobenzylthioinosine, indicating the presence of the equilibrative sensitive transporter rENT1.

Effect of hypertension on the integrity of blood brain and blood CSF barriers, cerebral blood flow and CSF secretion in the rat.

Hypertension has been related to the development of brain damage, dementia and other CNS dysfunctions. Disruption of the blood-brain barrier (BBB) is thought to contribute to these disorders. In this study, the integrity of both blood-brain and blood-CSF barriers during chronic hypertension was investigated. For this, the entry of [14C]sucrose and of lanthanum into brain tissue, choroid plexus, and CSF was studied. Also brain regional blood flow and brain [14C]sucrose volume of distribution were measured using indicator fractionation and ventriculo-cisternal perfusion methods, respectively. The above measurements were performed in normotensive (WKY) rats and in the spontaneously hypertensive rats (SHR). Choroid plexus and CSF uptakes of [14C]sucrose were found to be significantly greater in SHR compared to WKY rats (P<0.05). Intercellular entry of lanthanum was observed in choroidal tissue of SHR but not in that of WKY rats and at the BBB. Choroid plexus blood flow was significantly greater in SHR, 2.82+/-0.21 ml g(-1) min(-1), compared to 2.4+/-0.08 ml g(-1) min(-1) in WKY (P<0.05). There were no significant differences (P>0.05) in brain % water content and extracellular fluid [14C]sucrose volume of distribution between SHR and WKY rats. However, choroid plexus showed greater % water content in SHR (85.7+/-1.9%) compared to the WKY rats (81.5+/-1.7%). These results suggest that chronic hypertension in SHR may cause more pronounced defects in the integrity of the blood-CSF barrier than in the BBB.

Increased brain uptake and CSF clearance of 14C-glutamate in spontaneously hypertensive rats

Blood-to-brain and blood-to-CSF transport kinetics of 14C-glutamate in the spontaneously hypertensive rats (SHR) were studied using the in situ brain perfusion technique. Also, clearance of 14C-glutamate from CSF of SHR was studied using the ventriculo-cisternal (VC) perfusion technique. Blood-to-brain and blood-to-CSF transport kinetics showed greater rate of maximal transport into both brain and CSF of SHR compared to normotensive Wistar Kyoto (WKY) rats (p>0.05). Uptake into CSF of WKY and uptakes into brains of WKY and SHR did not show any significant diffusion (K(d)) of 14C-glutamate (p<0.05). However, some diffusion of 14C-glutamate only into CSF of SHR was observed, 0.031+0.006 microl min(-1) g(-1). Clearance of 14C-glutamate from CSF was greater in the SHR (28.33+/-6.9 microl min(-1)) compared to that in WKY rats (19.42+/-4.7 microl min(-1)). However, 14C-glutamate uptake by brain from CSF side was not significantly different between SHR and WKY rats (p>0.05). These results suggest that the greater blood-to-brain and blood-to-CSF entry of 14C-glutamate during hypertension may be balanced by greater removal of 14C-glutamate from CSF back to blood.

Transport of 14C-γ-aminobutyric acid into brain, cerebrospinal fluid and choroid plexus in neonatal and adult rats

In general blood to brain entry of amino acids is greater in the neonatal rats compared to the adults. gamma-Aminobutyric acid (GABA), a neurotransmitter amino acid, shows limited transport across the blood-brain barrier (BBB) in the adult rat. Characteristics of GABA entry into the immature rat brain is yet to be addressed. This investigation was set to study the entry of GABA into brain of the neonatal rat compared to the adult. Using the bilateral in situ brain perfusion technique, the entry of 14C-GABA into brain, cerebrospinal fluid (CSF) and lateral ventricles choroid plexuses was studied in the adult and neonatal rats. 14C-GABA uptake into neonatal rat brain after 20 min perfusion was 0.116+/-0.014 ml g(-1), approximately twice that of the adults (P 0.05), whereas maximal transport into the brain, V(max), was reduced from 0.152 to 0.068 nmol min(-1) g(-1) showing a significant reduction with age (P<0.05). In the neonate the entry of GABA into the CSF was dominant when compared to that into the brain, this could be due to a greater diffusional component, K(d), which was detected to be high in the neonate. In conclusion, the uptake of 14C-GABA into brain of the immature rats exceeded that in the adults which is thought to be due to both greater maximal transport and greater diffusion in the neonate compared to the adult.

NMDA preconditioning and neuroprotection in vivo: Delayed onset of kainic acid-induced neurodegeneration and c-Fos attenuation in CA3a neurons

Intraventricular (i.c.v.) kainic acid (KA) causes an acute excitotoxic lesion to the CA3 region of rodent hippocampus. Recent evidence implicated c-fos gene in regulating neuron survival and death following an excitotoxic insult. In this study we attempted to prevent KA-induced damage in CA3 neurons with NMDA preconditioning, which produced a marked expression of c-fos in the hippocampus. NMDA (0.6-6 microg, i.c.v.) was injected to anesthetized rats alone or 1 h before KA (0.15 microg, i.c.v.). Following KA injection, vibratome sections were processed for immunohistochemistry/electron microscopy. c-Fos and Nissl staining were used to estimate the extent of neuronal excitation and damage, respectively. Quantitative evaluation of c-Fos-labeled cells showed significantly less c-Fos in CA3a than in neighboring CA3b and CA2 from 1 to 4 h after KA alone. Attenuation of expressed c-Fos in CA3a was accompanied by damage of neurons with more apoptotic than necrotic signs. NMDA preconditioning elevated CA3a c-Fos expression and at 1 and 2 h exceeded markedly that after KA alone. However, at 4 h after KA, NMDA-preconditioned c-Fos induction in CA3a diminished to the same level as that seen after KA alone. The onset of neuronal degeneration was delayed in similar way. While NMDA-induced c-Fos expression in CA3a could be blocked by MK-801 completely, MK-801 and CNQX were both without significant effect on KA-induced c-Fos expression and neuronal damage. In conclusion, inhibition of c-Fos expression and onset of neuronal damage in CA3a following icv KA injection might be transiently delayed by i.c.v. NMDA preconditioning.

Time Course of Hyperosmolar Opening of the Blood-Brain and Blood-CSF Barriers in Spontaneously Hypertensive Rats

The time course of blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) responses to hyperosmolar mannitol infusion (HMI; 1.6 M) during chronic hypertension was investigated using 14C-sucrose as a marker of barrier integrity. 14C-sucrose entry into CSF of both spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats 2 min after HMI increased ∼7-fold compared to their respective control. The volume of distribution (Vd) of 14C-sucrose into brain cortex of SHR increased 13-fold 2 min after HMI while that in WKY rats increased only 4-fold. After HMI Vd of 14C-sucrose into the cortex of WKY, and CSF of both SHR and WKY remained steadily greater than their corresponding control for up to 30 min (p < 0.01), whereas in the cortex of SHR the Vd of 14C-sucrose reached control values 20 min after HMI (p > 0.05), indicating that after HMI the increase in paracellular diffusion of 14C-sucrose into SHR cortex was not persistent, in contrast to WKY rats and CSF of both SHR and WKY rats. Electron microscopy of the brain cortex after HMI showed capillary endothelial cell shrinkage and perivascular swellings in the brain cortex, and in the choroid plexus opening of tight junctions were observed. Our results indicate disruption of both the BBB and the BCSFB after HMI in both SHR and WKY rats. The disruption remained persistent up to 25 min after HMI at the BBB of WKY rats and BCSFB in both animal groups, while in SHR the protective function of the BBB returned to control values 20 min after HMI.

Changes in Acidic Amino Acid Efflux from Rat Brain with Maturation

Objective: Our previous studies have shown a greater uptake of acidic amino acids from the blood into the brain of neonatal when compared to that of adult rats. The aim of this study is to investigate whether a developmental change exists in the brain to blood efflux of this group of amino acids. Methods: The whole brain was perfused in situ with Ringer’s solution containing 14C-aspartate or 14C-glutamate for 10 min. The perfusion was then continued with 14C-free perfusate for a further 20 min and samples of jugular venous outflow were taken at 30-second intervals. The amount of radioactivity (in ln dpm) in each sample was then plotted against the sampling time, and the half-time (t½) for 14C-efflux was calculated. Results: Paper chromatography of the outflow samples revealed that more than 91% of the 14C-labelled acidic amino acid present in the effluent samples for up to 30 min of perfusion was chemically intact. The t½ for aspartate efflux in neonatal rats was 16.16 ± 0.76 min which was significantly slower (p < 0.05) than that for the adults, 10.06 ± 0.46 min (means ± SEM, n = 3 and 4). The t½ for glutamate efflux was also small in the adult brain where it was 50% the value seen in the neonates. Conclusion: The above results indicate that the systems involved in the efflux of acidic amino acids out of the brain favour retention and increased levels of this group of amino acids in the developing brain.