Kishena C. Wadhwani

Saturable Transport of Manganese(II) Across the Rat Blood‐Brain Barrier

Unanesthetized adult male rats were infused intravenously with solutions containing 54Mn (II) and one of six concentrations of stable Mn(II). The infusion was timed to produce a near constant [Mn] in plasma for up to 20 min. Plasma was collected serially and on termination of the experiment, samples of CSF, eight brain regions, and choroid plexus (CP) were obtained. Influx of Mn (JMn) was calculated from uptake of 54Mn into tissues and CSF at two different times. Plasma [Mn] was varied 1,000‐fold (0.076–78 nmol/ml). Over this plasma concentration range, JMn increased 123 times into CP, 18–120 times into brain, and 706 times into CSF. CP and brain JMn values fit saturation kinetics with Km (nmol/ml) equal to 15 for CP and 0.7–2.1 for brain, and Vmix (10–2 nmol · g−1· s−1) of 27 for CP and 0.025–0.054 for brain. Brain JMn except at cerebral cortex had a nonsaturable component. CSF JMn varied linearly with plasma [Mn]. These findings suggest that Mn transport into brain and CP is saturable, but transport into CSF is nonsaturable.

Identification of the cationic amino acid transporter (System y+) of the rat blood-brain barrier

Abstract: Cationic amino acids are transported from blood into brain by a saturable carrier at the blood‐brain barrier (BBB). The transport properties of this carrier were examined in the rat using an in situ brain perfusion technique. Influx into brain via this system was found to be sodium independent and followed Michaelis‐Men‐ten kinetics with half‐saturation constants (Km) of 50–100 μM and maximal transport rates of 22–26 nmol/min/g for L‐lysine, L‐arginine, and L‐ornithine. The kinetic properties matched that of System y+, the sodium‐independent cationic amino acid transporter, the cDNA for which has been cloned from the mouse. To determine if the cloned receptor is expressed at the BBB, we assayed RNA from rat cerebral microvessels and choroid plexus for the presence of the cloned transporter mRNA by RNase protection. The mRNA was present in both cerebral microvessels and choroid plexus and was enriched in microvessels 38‐fold as compared with whole brain. The results indicate that System y+ is present at the BBB and that its mRNA is more densely expressed at cerebral microvessels than in whole brain.

β-amyloid increases choline conductance of PC12 cells: possible mechanism of toxicity in Alzheimer's disease

Abstract When β-amyloid-(1–40) is added to PC12 cells, there is an increase in choline conductance that is proportional to the β-amyloid concentration. If a similar effect occurs in cholinergic brain cells of Alzheimers disease patients, the intracellular choline concentration would be reduced, leading to a decrease in the production of acetylcholine. This could explain the reduced level of acetylcholine that has been found in post-mortem brain tissue of Alzheimers disease patients.

β-Amyloid polypeptide increases calcium-uptake in PC12 cells : a possible mechanism for its cellular toxicity in Alzheimer's disease

Calcium-uptake into PC12 cells was measured by incubation with45Ca after the cells were exposed for 24 h to β-amyloid peptide(1–40) at concentrations between 0 and 46 μM. The rate of influx of45Ca into PC12 cells was constant for the first 10 min. For 46 μM β-amyloid peptide(1–40), the rate of influx was about 1,300 ions/s/μm2 and the number of cells decreased significantly. There was no significant decrease in cell number when cells were exposed to β-amyloid in calcium-free medium. These results indicate that β-amyloid increases calcium uptake into PC12 cells, and suggest that the increased uptake is responsible for the toxicity of β-amyloid in PC12 cells.

Blood-nerve and blood-brain barrier permeabilities and nerve vascular space in Fischer-344 rats of different ages

The permeability-surface area product (PA) of [3H]- or [14C]sucrose at the blood-nerve barrier (BNB) of the sciatic nerve; and at the blood-brain barrier (BBB), were determined in Fischer-344 rats at 3, 11 and 31 months of age. PA was determined by using an in vivo i.v. bolus injection of radiotracer with two-time point graphic and quantitative autoradiographic methods. Vascular space and water content of the tibial nerve of these rats also were determined using quantitative morphometry and dry and wet weight ratios, respectively. There was no significant difference between mean PA(BNB) in any age group [(PA(BNB) at 3 months = 1.2 +/- 0.1 (mean +/- S.E.), at 11 months = 1.8 +/- 0.3; and at 31 months = 1.4 +/- 0.2 x 10(5) ml/s . g wet wt; n = 5-8 rats], nor any difference in PA(BBB). The mean ratio (%) of surface area of endoneurial blood vessels/nerve cross-section of the tibial nerve also did not differ between any group [3 months: 16 +/- 2 vessels; mean surface area ratios = 2.20 +/- 0.10%, n = 5; 11 months: 22 +/- 3 vessels and 2.48 +/- 0.21%, n = 5; 11 months: 22 +/- 3 vessels and 2.48 +/- 0.21%, n = 5; and at 31 months: 26 +/- 1 vessels and 2.40 +/- 0.23%, n = 4). The mean nerve water in rats at 31 months was 64.8 +/- 1.1% wet wt and did not differ from that at 11 months (66.0 +/- 0.6% wet wt) or at 3 months (65.1 +/- 1.0% wet wt) (n = 5-8 nerves). Our results indicate that BBB and BNB integrities are not altered in senescent Fischer-344 rats.

Prevention of nerve edema and increased blood-nerve barrier permeability-surface area product in galactosemic rats by aldose reductase or thromboxane synthetase inhibitors.

Nerve water content and the permeability–surface area product (PA) to [3H]- or [14C]sucrose at the blood-nerve barrier were determined in unanesthetized control rats fed a normal diet and in rats fed galactose with or without an aldose reductase inhibitor (Statil or AL 1576) or a thromboxane synthetase inhibitor (CGS 12970). Nerve water content was determined by taking the difference between dry and wet weights of whole tibial nerves. PA was determined by an intravenous bolus injection of radiotracer with multiple–time-point graphic and quantitative autoradiographic methods. The mean nerve water content in galactosemic rats was 15% higher than in control rats after 7–11 mo on the diet. Statil and AL 1576 prevented nerve edema, but CGS 12970 was only partially effective in preventing an increase in nerve water content in galactose-fed rats. In galactosemic rats, the mean PA to sucrose at the blood-nerve barrier, calculated from nerve dry weight, was twofold higher than in control rats. Treatment with Statil, AL 1576, or CGS 12970 prevented increased PA. Our results suggest that nerve edema and increased blood-nerve barrier PA are secondary to polyol production and can be prevented by inhibiting aldose reductase.

Adrenergic innervation in the tibial and vagus nerves of rats with streptozotocin-induced diabetes

Adrenergic innervation of tibial and vagus nerves was studied after 1-16 weeks duration of streptozotocin (STZ)-induced diabetes in rats. Sucrose-phosphate glyoxylic acid (SPG) histochemistry and the formaldehyde-induced fluorescence (FIF) method were used to demonstrate adrenergic nerve fibers in the epi-perineurial and endoneurial compartments. Densities of innervation were quantitated with fluorescence microscopy. The density of periarteriolar adrenergic innervation in the epi-perineurium of the tibial and vagus nerves was increased 5 and 12 weeks after STZ injections as compared with control. At 16 weeks, mean densities of periarteriolar innervation in epi-perineurium had returned to or below control levels in both nerve types. In the endoneurium, however, the mean density of adrenergic nerve fibers decreased gradually at 5 weeks after induction of diabetes in both nerves, and was totally absent at 12 weeks. At 16 weeks no sign of recovering innervation in the endoneurium was seen. In conclusion, adrenergic innervation goes through similar pathological alterations both in tibial and vagus nerves shortly after the induction of streptozotocin diabetes. These changes may contribute to diabetic peripheral neuropathy by impairing the regulation of nerve blood flow.

Perineurial permeability and endoneurial edema during Wallerian degeneration of the frog peripheral nerve

Perineurial permeabilities to [3H]sucrose and [14C]dextran (MW = 70,000), and water content, conduction velocity (CV) and maximum amplitude (MAP) of the compound action potential, were determined in Wallerian degenerated nerves (sciatic or tibial) of the frog and compared with values in the contralateral uncut nerves. Three days after transection of the lumbosacral plexuses, about 2 cm proximal to the sciatic nerve, mean water content of the sciatic nerve was significantly higher than in the contralateral uncut nerve. After 10 days, the degenerating sciatic nerve showed significant increases in the mean perineurial permeabilities to [3H]sucrose and [14C]dextran when compared to values in the contralateral nerve. Means MAPs and CVs were significantly decreased. At 21 days and after, no compound action potential was detected and perineurial permeability and nerve water content had increased further. Decreases in mean MAPs and CVs and permeability increases of the perineurium were less in degenerating tibial nerves than in degenerating sciatic nerves. It is concluded that following transection, (1) Wallerian degeneration produces an irreversible increase in perineurial permeability, (2) the increase of perineurial permeability follows a proximodistal gradient, and (3) the frog peripheral nerve develops endoneurial edema during Wallerian degeneration as do degenerated nerves of mammals.

Calcium transfer at the blood-nerve barrier of the frog sciatic nerve

Calcium transfer across the blood-nerve barrier of the frog sciatic nerve was studied using an in situ perfusion technique and an in vivo i.v. bolus injection technique. The permeability-surface area product of 45Ca at the blood-nerve barrier, (PA)BNB, calculated from radioactivity in the desheathed nerve segment after 5 min of circulation of tracer, and corrected for the residual radioactivity in the blood space, equaled 4.4 +/- 0.4 (S.E.M.) X 10(-5) ml.s-1.g-1 wet wt. The (PA)BNB of 45Ca was independent of [Ca2+] in the perfusion medium between 0.18 and 18 mM. The permeability-surface area products of 45Ca across the perineurium [(PA)per] also was measured by an in situ incubation technique, and equaled 1.45 +/- 0.41 X 10(-5) ml.s-1.g-1 wet wt. (n = 8). The half time (t 1/2) for nerve calcium to equilibrate with plasma calcium was calculated to be 60 min. The low, passive permeability to calcium of the blood-nerve barrier probably limits marked calcium concentration changes in nerve endoneurium following transient changes of plasma calcium, but should not alter steady-state responses.

Regeneration of perivascular adrenergic innervation in rat tibial nerve after nerve crush

SummaryAdrenergic innervation of blood vessels in the rat tibial nerve during degeneration and regeneration was studied using the formaldehyde-induced fluorescence method. The left sciatic nerve was crushed with suture threads to produce a 4-mm length of crushed nerve. At 1, 3, 7, 14, 28, 56 and 84 days after nerve crush, degenerative and regenerative changes in the nerve were verified using light microscopy. At each time point, adrenergic innervation was examined in epi-perincurial whole mount and nerve cross-section preparations. One day after nerve crush, fluorescence of adrenergic nerve fibers in the endoneurium was absent. Fluorescent adrenergic nerve fibers reappeared in the endoneurium at day 56 and reached the control density by 84 days. In the epi-perineurium, adrenergic innervation of small and medium-size arterioles was absent at 3 days, in large arterioles at 7 days. At 56 days, all epi-perineurial arterioles were reinnervated by a faint, sparse adrenergic network, which reached the control density at 84 days. The results suggest that adrenergic innvervation in the rat peripheral nerve is lost during nerve degeneration, but recovers when the nerve has regenerated.