Iris vasculopathy in galactose-fed rats.

Increased iris vessel permeability observed in diabetics has also been reported to occur in diabetic animals and galactose-fed rats. The potential role of aldose reductase in the induction of iris vessel changes has been investigated in rats fed a 50% galactose diet with/without the aldose reductase inhibitors AL 1576, sorbinil or ponalrestat for 7 to 18 months. Compared to normal control rats, long-term galactose-fed rats display a breakdown of the blood-aqueous barrier due to iris vessel changes that include focal straightening, dilation, constriction, increased permeability, ischemia and new vessel proliferation. The onset and progression of these iridal vessel changes were prevented by the aldose reductase inhibitors AL 1576 and sorbinil, and reduced by Ponalrestat. Computerized analyses of lumen areas of iris vessels indicated an 18-fold decrease in the vascular area near the pupillary boarder in untreated galactose-fed rats compared with age-matched controls and galactose-fed rats treated with aldose reductase inhibitors. These observations linking iris vessel changes with galactose-feeding, coupled with the fact that aldose reductase inhibitors also prevent these changes, strongly suggest a link between the sorbitol pathway and the appearance and progression of iris vessel changes.

Permeability of the blood-retinal and blood-aqueous barriers in galactose-fed rats.

Blood-retinal barrier (BRB) and blood-aqueous barrier (BAB) permeability were assessed by various methods to clarify conflicting reports on whether blood-retinal barrier permeability changes occur in diabetic and galactose-fed rats and to assess the potential role of aldose reductase in this process. Different molecular weight probes were utilized in rats fed for 7-11 months either a normal diet or a diet containing 50% galactose with/without the aldose reductase inhibitor AI1576 or Ponalrestat. BRB and BAB were assessed through radiolabelled sucrose permeability studies in eyes where the anterior segment was frozen during dissection compared to eyes where the anterior segments were not frozen and by quantitative autoradiography. In addition, histological studies using Evans Blue dye, microperoxidase and horseradish peroxidase were conducted. In untreated galactose-fed rats a 4-fold increase in the mean permeability surface area product (PA) to sucrose at the BRB was observed when the aqueous humor was not frozen during retinal dissection. However, no increase in the mean PA to sucrose was observed when the aqueous humor of similar eyes was frozen during dissection. Similarly, no retinal vessel permeability increase was observed by either quantitative autoradiography of [3H]-sucrose after 15 minutes of circulation or histological studies with microperoxidase, horseradish peroxidase or Evans Blue dye. Examination of the BAB revealed an increase in the permeability of iris vessels but not the ciliary body in galactose-fed rats which was reduced by treatment with the aldose reductase inhibitor AI1576. These data indicate that while BRB permeability is not increased in galactose-fed rats, BRB permeability measurements with isotopes are subject to possible contamination from the aqueous humor in untreated galactose-fed rats, which can result in false observations of increased BRB permeability.

beta-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 with 45Ca after the cells were exposed for 24 h to beta-amyloid peptide(1-40) at concentrations between 0 and 46 microM. The rate of influx of 45Ca into PC12 cells was constant for the first 10 min. For 46 microM beta-amyloid peptide(1-40), the rate of influx was about 1,300 ions/s/microns 2 and the number of cells decreased significantly. There was no significant decrease in cell number when cells were exposed to beta-amyloid in calcium-free medium. These results indicate that beta-amyloid increases calcium uptake into PC12 cells, and suggest that the increased uptake is responsible for the toxicity of beta-amyloid in PC12 cells.

beta-Amyloid increases choline conductance of PC12 cells: possible mechanism of toxicity in Alzheimer's disease.

When beta-amyloid-(1-40) is added to PC12 cells, there is an increase in choline conductance that is proportional to the beta-amyloid concentration. If a similar effect occurs in cholinergic brain cells of Alzheimer's 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 Alzheimer's disease patients.

Quantitative reverse transcriptase-polymerase chain reaction of glucose transporter 1 mRNA levels in rat brain microvessels.

We investigated the usefulness of reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify glucose transporter 1 (GLUT1) mRNA in cerebral microvessels. The technique was validated using an in vitro-transcribed RNA fragment (riboprobe) of partial 3' noncoding sequence of rat brain GLUT1 gene. A known amount of the riboprobe was reverse-transcribed to cDNA (target DNA). PCR primers were made to amplify a 292-bp fragment of the target DNA. The 5' primer was end labeled with 32P. An oligonucleotide of 100 bp containing the same sequences as the first 30 and the last 70 bases of the 292-bp fragment of the target DNA was synthesized and used as competitive DNA. The target DNA was coamplified with increasing amounts of competitive DNA using the same two primers. The ratio of radioactivity between amplified products of the target DNA (292-bp fragment) and the competitive DNA (100-bp fragment) was determined quantitatively after separation by gel electrophoresis and radioactivity counting. This method gave an accurate estimation of the amount of the riboprobe in the reaction and a 2- to 5-fold change in the amounts could be detected. By this method, the mean amount of GLUT1 mRNA from purified rat brain microvessels was estimated to be 1.5 +/- 0.1 x 10(-6) ng/ng total RNA. This value was about 10-fold higher than that in rat cell line PC12.

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

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-Menten kinetics with half-saturation constants (Km) of 50-100 microM 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.

Enhanced permeabilities of cationized-bovine serum albumins at the blood-nerve and blood-brain barriers in awake rats.

Permeability-surface area products (PAs) of the blood-nerve barrier (BNB) and blood-brain barrier (BBB) to 125I-labeled native bovine serum albumin (nBSA, pI approximately 4), and to 2 cationized albumins (cBSA) of differing pI (pI approximately 8 and 11), were quantitatively determined in awake rats, using an i.v. bolus injection technique. Mean PAs of the BNB and BBB to 125I-nBSA, after a circulation time of up to 120 min, were (0.17 +/- 0.23) and (0.09 +/- 0.05) x 10(-5) ml/s.g. wet wt, respectively (n = 12 rats), and were not significantly different from 0 (P greater than 0.05). Mean PAs of the BNB and BBB to 125I-cBSA (pI approximately 8), after circulation time of 12 min, were (1.9 +/- 0.1) and (1.7 +/- 0.1) x 10(-5) ml/s.g wet wt, respectively (n = 8). Significant greater PAs, at both the BNB and BBB to 125I-cBSA (pI approximately 11) [(8.2 +/- 1.8) and (3.0 +/- 0.6) x 10(-5) ml/s.g wet wt, respectively (n = 12)], than both PA's of nBSA and cBSA (pI approximately 8) were found. The accumulation of 125I-cBSA in epi-perineurial tissues also was higher than that of 125I-nBSA, and was related to the degree of cationization. Our results indicate that, as at the BBB, the transfer of cationized serum albumin is enhanced over that of native albumin at the BNB of the mammalian peripheral nerve.

Saturable transport of manganese(II) across blood-nerve barrier of rat peripheral nerve.

To determine whether the blood-nerve barrier of the rat peripheral nerve transports manganese(II) (Mn) by a saturable mechanism similar to that found at the blood-brain barrier, we measured the uptake of 54Mn from blood into desheathed sciatic nerve and into cerebral cortex of awake rats at different plasma concentrations of unlabeled Mn using an intravenous infusion technique. The unidirectional influx (Jin) of Mn into sciatic nerve was facilitated and saturable, when steady-state plasma Mn ranged from 4 to 4,312 ng/ml (0.073-78.4 microM), as was the unidirectional influx of Mn into the cerebral cortex. Michaelis-Menten constants (Km and Vmax) and the passive diffusion constant (Kd), determined by nonlinear least squares, were as follows: for the blood-nerve barrier (sciatic nerve) Km = 4.7 microM, Vmax = 0.56 x 10(-3) nmol.s-1.g wet wt-1, and Kd = 6.3 x 10(-6) ml.s-1.g wet wt-1; for the blood-brain barrier (cerebral cortex) Km = 1.0 microM, Vmax = 0.40 x 10(-3) nmol.s-1.g wet wt-1, and Kd = 0.3 x 10(-6) ml.s-1.g wet wt-1. The results demonstrate facilitated concentration-dependent mechanisms of transport of Mn at the blood-nerve and blood-brain barriers.

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 Vmax (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.

Regional blood-brain barrier transport of cationized bovine serum albumin in awake rats.

Regional blood-brain barrier permeability-surface area products (PAs) of cationized bovine serum albumin (cBSA) with isoelectric point (pI) approximately 8 or greater than or equal to 11 and of native bovine serum albumin (nBSA;pI approximately 4) were determined in awake male Sprague-Dawley rats after bolus intravenous injection. The albumins were labeled with 125I. Brain uptakes were assessed by autoradiography and by direct assay of radioactivity in brain regions. nBSA uptake into brain was statistically insignificant over 3 h, whereas cBSA uptake was significantly even at 6 min. Mean PA values of cBSA with pI approximately 11 (1.69-2.65 x 10(-5) ml.s-1.g-1) in most brain regions were twofold higher than PAs of cBSA with pI approximately 8 (0.98-1.37 x 10(-5) ml.s-1.g-1), whereas mean PA for nBSA did not differ significantly from zero. Autoradiographs of brain slices and net distributions in brain compartments at 6 and 30 min after injection suggested that cBSA entered the brain parenchyma via blood vessels and cerebrospinal fluid but that the former was the main route. The results quantitate for the first time regional brain PA values for cationized proteins and suggest specific mechanisms at cerebral blood vessels that distinguish transport of cationized from noncationized macromolecules.

Blood-nerve barrier in the frog during wallerian degeneration: are axons necessary for maintenance of barrier function?

Blood-nerve barrier tissues (endoneurial blood vessels and perineurium) of the frog's sciatic nerve were studied during chronic Wallerian degeneration to determine whether barrier function depends on the presence of intact axons. Sciatic nerves of adult frogs were transected in the abdominal cavity; the ends were tied to prevent regeneration and the distal nerve stumps were examined. Vascular permeabilities to horseradish peroxidase and to [14C]sucrose increased to day 14, returned toward normal levels by 6 weeks, and continued at near normal levels to 9 months. Perineurial permeabilities to the tracers increased by day 10 and remained elevated at 9 months. Proliferation of perineurial, endothelial, and mast cells occurred between 3 days and 6 weeks, resulting in an increased vascular space (measured with [3H]dextran) and number of vascular profiles. The perineurium increased in thickness and the mast cells increased in number. This study indicates that during Wallerian degeneration of the frog's sciatic nerve there is 1) a transitory increase in vascular permeability distal to the lesion, that is related to changes within the endoneurium; 2) an irreversible increase in permeability of the perineurium, which begins later than that seen in the endoneurial blood vessels; and 3) proliferation of non-neuronal components in the absence of regenerating neuronal elements. The results indicate that maintenance of vascular integrity does not require the presence of axons in the frog's peripheral nerve, whereas perineurial integrity and barrier function are affected irreversibly by Wallerian degeneration.

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.

Transfer of 45Ca and 36Cl at the blood-nerve barrier of the sciatic nerve in rats fed low or high calcium diets.

Unidirectional fluxes of 45Ca, 36Cl, and of [3H]mannitol from blood into the sciatic nerve and cerebral cortex were determined from 5- and 15-min uptakes of these tracers after an intravenous (i.v.) bolus injection in awake rats. Rats were fed diets for 8 wk, that had either a low (0.01% wt/wt), normal (0.67%), or high (3%) Ca content. Plasma [Ca] was 32% less and 11% more in rats fed low (LOCA) and high Ca diets (HICA), respectively, than in rats fed a normal Ca diet (CONT). The mean permeability-surface area product (PA) of 45Ca at the blood-nerve barrier was about eightfold higher than at the blood-brain barrier in the same animals and did not differ significantly between groups (greater than 0.05). Mean PA ratios of 45Ca/36Cl for the blood-nerve and blood-brain barriers in CONT rats, 0.52 +/- 0.04 and 0.40 +/- 0.02, respectively, were not significantly different from corresponding ratios in LOCA and HICA groups, and corresponded to the aqueous limiting diffusion ratio (0.45). Our results show no evidence for concentration-dependent transport of Ca over a plasma [Ca] range of 0.8-1.4 mmol/liter at the blood-nerve barrier of the rat peripheral nerve, and suggest that Ca and Cl exchange slowly between nerve and blood via paracellular pathways.

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

Adrenergic 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-perineurial 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 innervation in the rat peripheral nerve is lost during nerve degeneration, but recovers when the nerve has regenerated.

Postnatal development of adrenergic innervation in the tibial and vagus nerves of Fischer-344 rats.

Adrenergic innervation of rat tibial and vagus nerves was studied in male Fischer-344 rats between 1 and 84 days of age, using sucrose-phosphate-glyoxylic acid (SPG) histochemistry and the formaldehyde-induced fluorescence (FIF) method. Adrenergic nerve fibers were found in epi-perineurial blood vessels of the vagus nerve at one day of age, whereas blood vessels in the tibial nerve received the first adrenergic nerve fibers at 3 days. A few adrenergic nerve fibers were seen in the endoneurium of both tibial and vagus nerves at 7 days. The densities of adrenergic innervation increased gradually during the first 4 postnatal weeks, and at 21 days the distributions of adrenergic innervation in both nerves resembled those in adult animals. The results suggest that development of adult adrenergic innervation in rat peripheral nerves occurs during the first postnatal month and that sympathetic innervation becomes available to regulate nerve blood flow within this period.

Facilitated transport of L-phenylalanine across blood-nerve barrier of rat peripheral nerve.

L-Phenylalanine transport across the blood-nerve barrier was studied by perfusing Ringer solution containing trace amounts of L-[14C]phenylalanine and [3H]dextran (a vascular marker) through the hindlimb of an anesthetized rat. At the end of perfusion, a segment of sciatic nerve was removed, frozen, desheathed, and processed for radioactivity counting. The permeability-surface area product of the blood-nerve barrier (PABNB) to L-[14C]phenylalanine decreased from 58 +/- 9 X 10(-5) to 1.7 +/- 0.3 X 10(-5) (SE) ml.s-1.g wet wt-1, as the perfusate concentration of unlabeled L-phenylalanine was increased to 10 mM. D-Phenylalanine had less of an inhibitory effect on L-[14C]phenylalanine uptake than did L-phenylalanine. PABNB did not change in the presence of 50 mM alpha-(methylamino)isobutyric acid (MeAIB) in the perfusion medium, nor by replacing NaCl by Tris.HCl. However, addition of 50 mM 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) reduced PABNB to 2.1 +/- 0.3 X 10(-5) ml.s-1.g wet wt-1 (n = 4). PA at epiperineurial barrier (PAper) of L-[14C]phenylalanine at the epiperineurial barrier, determined from in situ incubation, was 1.4 +/- 0.2 X 10(-5) ml.s-1.g wet wt-1 (n = 6) and was unaffected by 10 mM L-phenylalanine concentration in the incubation medium. The results demonstrate for the first time carrier-mediated transport system for L-phenylalanine at the blood-nerve barrier of the rat peripheral nerve.

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.

Adrenergic innervation of the tibial and vagus nerves in rats of different ages.

Adrenergic nerve fibers innervating blood vessels in the epi-perineurium and endoneurium of the tibial and vagus nerves of male Fischer-344 rats of different ages were examined using formaldehyde-induced fluorescence technique and fluorescence microscopy. Between 6 and 24 months of age, no significant difference in the mean density of perivascular innervation in the epi-perineurium of the nerves was found, whereas between 24 months and 30 months the density decreased in both nerve types. The intensity of noradrenaline fluorescence of nerve fibers also was decreased in the oldest age group. In the endoneurium, the density of adrenergic nerve fibers was reduced by 57% at 24 months, and by more than 98% at 30 months as compared with 18 months. Age-related differences were similar in the tibial and vagus nerves. The results suggest that adrenergic neuronal control of the microcirculation in the rat tibial and vagus nerves is lost during aging as it is in other organs of the cardiovascular system.