AIB Transfer Research Articles

A Quantitative Spatial Analysis of the Blood–Spinal Cord Barrier: I. Permeability Changes after Experimental Spinal Contusion Injury

Blood–spinal cord barrier (BSB) permeability was measured using quantitative autoradiography following contusion injury to the rat spinal cord. Permeability was assessed by calculating blood-to-tissue transfer constants (Kivalues) for the vascular tracer [14C]-α-aminoisobutyric acid (AIB) in injured (3, 7, 14, and 28 days postinjury), laminectomy control, and uninjured control animals. Permeability was quantitated using four separate imaging techniques in gray and white matter throughout the rostro-caudal extents of the forming lesion. Away from the epicenter, gray matter permeability was further differentiated within discrete spinal lamina using computerized templates. Regardless of the type of analysis used, increased AIB permeability (Kivalues) was noted at all survival times in all tissue regions with respect to both uninjured and laminectomy control groups. The data indicate a large increase in individualKivalues throughout the dorsoventral axis of the spinal cord at 3 days postinjury (≈6–9 ml/kg/min). By 7 days,Kivalues were quantitatively smaller (≈4–5 ml/kg/min) in all regions compared with 3-day tissues. Despite further attenuation of AIB uptake in the gray matter at 14 and 28 days postinjury, circumferential white matter tracts showed a secondary increase in permeability compared to 7-day tissue. Permeability in the white matter at 14–28 days postinjury (≈5–6 ml/kg/min) was comparable to that at 3 days postinjury (6–7 ml/kg/min). Measurements of the axial distribution of AIB permeability indicate increased BSB permeability over several segments rostral and caudal to the lesion epicenter (≈3 cm in both directions). Secondary elevations of AIB transfer in the spinal white matter between 14 and 28 days were colocalized with zones of immunohistochemically defined microglial clusters. The known plasticity of this cell type in response to changes in the extracellular microenvironment suggests that the spinal white matter at later survival times (14–28 days postinjury) is an area of dynamic vascular and/or axonal reconstruction. The implications of increased permeability to both tissue injury and neural regeneration are discussed.

A Quantitative Spatial Analysis of the Blood–Spinal Cord Barrier: II. Permeability after Intraspinal Fetal Transplantation

In previous experiments we utilized quantitative autoradiography to temporally describe vascular permeability of a radiolabeled vascular tracer following spinal contusion injury in the rat. In the present report we compare these findings with permeability assessments following fetal grafting in the contused rat spinal cord. At 10 days postinjury, Embryonic Day 14 spinal tissue was grafted into the lesioned spinal cord of Sprague–Dawley rats. At 7, 14, and 28 days postgrafting the α-aminoisobutyric acid (AIB) technique was used to assess blood-to-tissue transfer rates in graft and host tissue over several segments of the injured spinal cord. Regional changes in permeability were assessed using four distinct image analysis techniques. Using these methods, we have previously shown that contusion injury alone results in a chronic relapse in vascular permeability. The present data indicate that fetal transplants at 7 days postgrafting have AIB transfer rates that are significantly above uninjured control levels and are similar in magnitude to neighboring host spinal tissue. In addition, permeability in 14- and 28-day intraspinal grafts decreased relative to that of the 7-day transplant group, but remained significantly elevated at and rostral to the injury epicenter. Alternately, graft and host tissue in regions caudal to the injury epicenter (e.g., T10–L2) acquired a functional barrier to AIB as early as 14 days posttransplantation. These experiments suggest that graft development occurs in a different manner or at a different rate in segments of the injured spinal cord rostral and caudal to the injury site. Additionally, it appears that vascular permeability of the injured spinal cord can be influenced by the process of intraspinal transplantation.

Calcium dependence of alpha-aminoisobutyric acid transfer in human bilaterally perfused placental lobules.

Human isolated bilaterally perfused placental lobules were employed to study the Ca++ dependence of materno-fetal transfer of the non-metabolisable amino acid, alpha-aminoisobutyric acid (AIB). Bilateral perfusion with Ca(++)-free Krebs' solution containing 2 mmol/l EDTA for 60 min resulted in a significant reduction in materno-fetal AIB transfer compared to controls perfused with normal Krebs' solution. However, bilateral perfusion with Krebs' solution containing: 2.5 mmol/l Ca++ and 2 mmol/l EDTA; 3.8 mmol/l Ca++ with no EDTA; or Ca(++)-free Krebs' solution alone did not produce significantly different materno-fetal AIB transfer compared to placentae perfused with normal Krebs' solution. The reduction in AIB transfer during perfusion with Ca(++)-free Krebs' solution with EDTA was partially restored with subsequent reperfusion with normal Krebs' solution for 90 min. These results suggest that AIB transfer in human perfused placental lobules is at least partially Ca(++)-dependent.

Early and transient increase of rat hippocampal blood-brain barrier permeability to amino acids during kainic acid-induced seizures

The effects of kainic acid (KA) induced seizures on the permeability of hippocampal blood-brain barrier (BBB) to amino acids were investigated using a new method based on the combination of brain microdialysis of α-[ 3H]aminoisobutyric acid (AIB) and blood sampling. The transfer of AIB through the BBB was thus, for the first time, monitored in vivo in awake animals. KA administration i.v. produces a rapid and transient increase in hippocampal transfer of AIB after the beginning of seizures which is closely correlated to epileptic activity. The possible consequences of this increased transport of AIB from blood to brain are discussed in relation to previously reported transient augmentations of extracellular cerebral glutamate concentrations during KA-induced epilepsy.

Efferent microvascular responses to electrical stimulation of the area postrema in rats

As part of its role to transduce blood-borne and afferent neural stimuli to the brain, the area postrema conducts efferent projections monosynaptically to individual nuclei of the medulla oblongata and pons. We hypothesized that electrical activation of the area postrema would mimic this transduction process and couple microvascular responses in efferent sites to local increases in tissue metabolism reported previously. We used quantitative autoradiographic techniques and image analysis to measure capillary transfer constants for [ 14C]α-aminoisobutyric acid (AIB, a small, neutral amino acid) and blood flow (iodo[ 14C]antipyrine) in individual brainstem structures of anesthetized rats. The area postrema was stimulated electrically by means of a monopolar microelectrode positioned stereotaxically 100 μm deep in the dorsocentral aspect of the organ. There were no significant effects of stimulation on [ 14C]AIB influx or blood flow in control hindbrain structures where postremal projections are sparse or absent — the spinal trigeminal nucleus, reticular formation, or cerebellar vermis. Stimulation of the area postrema produced equivalent increases in transcapillary influx of [ 14C]AIB and capillary blood flow in the nucleus of the solitary tract, dorsal motor nuclei of the vagus nerves, ventrolateral medullary C1 region, locus coeruleus, dorsal tegmental nuclei, and lateral parabranchial nuclei. Formation of ratios interrelating rates of [ 14C]AIB influx and blood flow with previously assessed values of tissue glucose metabolism indicated that these measures increased proportionately during postremal stimulation. Such proportional increases in capillary [ 14C]AIB transfer and blood flow during tissue activation by area postrema stimulation are consistent with interpretation that the increase in blood flow resulted from recruitment of unused surface area in the capillary networks of individual efferent nuclei. Specifically in the ventrolateral medullary C1 region, the increase in blood flow significantly exceeded that of tissue metabolic rate, an outcome that may indicate intrinsic neurogenic dilatation of C1 vessels by area postrema projections.

Regional quantitative permeability of blood-brain barrier lesions in rats with chronic renal hypertension

Blood-brain transfer constants for a small, neutral amino acid tracer, [ 14C]α-aminoisobutyric acid (AIB), were measured by quantitative autoradiography and image analysis in 15 individual brain structures of 2-kidney, 1-clip renal hypertensive rats (RHR) and age-matched normotensive cotrols (NR). Mean arterial presures (MAP) for 4 month-old RHR and NR were 182 ± 19 and 121 ± 3mmHg, respectively. Most brain structures in RHR had very low [ 14C]AIB transfer constants similar to those in NR (1–3 μl·g −1·min −1), indicate of normal blood-brain barrier (BBB) function. Focal lesions, however, having transfer contants 2–7x normal and measuring less than 1.7 mm 2 in area, appeared in RHR primarily in the cerebellar vermian and cerebral cortices. Chronic unilateral cervical sympathectomy did not influence the incidence or magnitude of BBB lesions in the denervated hemisphere of RHR. Acute arterial hypertension produced by systemic infusion of phenylephrine (elevation of MAP in RHR by 43%) increased the incidence and magnitude of lesions by 48% and 2–12x, respectively, although many brain regions in acutely hypertensive RHR retained normal permeability to [ 14C]AIB. The results demonstrate normal BBB permeability for much of the brain in chronic renal hypertension, with focal lesions having 7x or less the normal rate of bloodbrain transfer for a small physiological probe.

Effect of inorganic mercury on in vitro placental nutrient transfer and oxygen consumption

The effect of mercury (HgCl 2) on placental amino acid and glucose transfer as determined by the use of their nonmetabolizable radioactive analogues, aminoisobutyric acid (AIB) and 3- o-metyhl glucose (3MG), respectively, was studied in an in vitro perfusion model of a term human placenta. Hg 2+ was found to decrease the transfer and accumulation of AIB without affecting 3MG transfer. It was also found to decrease the placental oxygen consumption rate. Placental circulation and tissue morphology remained intact, as demonstrated by the antipyrine transfer rate, and by electron microscopy, respectively. The mechanism by which Hg 2+ may interfere with placental amino acid transfer and accumulation is discussed. Although much higher concentrations than those found in the ordinary polluted environment were used, this is the first report showing that Hg 2+ interferes with an essential human placental function in a system employing a whole human placental cotyledon. This finding may indicate the possible involvement of Hg 2+ in impaired organogenesis in early pregnancy or deranged fetal growth during the last trimester.

Acetylcholine output and materno-fetal alpha-aminoisobutyric acid transfer in the perfused human placental lobule

Dually perfused human placental lobules were employed to study the effects of vesamicol (an inhibitor of acetylcholine (ACh) storage) and physostigmine (an inhibitor of cholinesterase breakdown of ACh) on ACh output from fetal vessels and on materno-fetal transfer of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB). Bilateral perfusion with vesamicol (100 mumol L-1) or omission of physostigmine (2 x 7 mumol L-1) from the perfusate significantly reduced the output of ACh. However, neither drug had any significant effect on AIB transfer. Thus, the results of this study do not provide evidence for a close relationship between the placental cholinergic system and materno-fetal AIB transfer.

Cerebrovascular permeability in intracerebral implants of foetal basal forebrain cell suspensions

Blood-brain barrier permeability was measured using [14C]-labelled a-aminoisobutyric acid (AIB) quantitative auto-radiography in rats which had previously received unilateral ibotenate-induced lesions of the nucleus basalis followed by intracortical implantation of foetal basal forebrain cell suspensions. The permeability characteristics of intracortical transplants were found to be dependent upon the site of implantation. Superficial transplants were invariably associated with AIB transfer constants (Ki) 3- to 4-fold higher than those in corresponding contralateral host cortex. In transplants sited deep in host neocortex, Ki values were not significantly different from those measured in surrounding host brain tissue.

Placental Transfer of Glucose and Amino Acids in Intrauterine Growth Retardation: Studies with Substrate Analogs in the Awake Guinea Pig

After experimentally inducing growth retardation by unilateral uterine artery ligation in midpregnancy, placental blood flow (PBF) (microsphere technique) and placental transfer of 14C-aminoisobutyric acid (AIB) and 3H-methylglucose (MG) were studied at d 44, 50, or 63 of gestation in 19 chronically catheterized awake guinea pigs. At d 44, fetal wt (FW) and placental wt were reduced by 16 and 18%, respectively, in the ligated horn and FW/PBF was increased by 122%. Placental efficiency to transfer MG and AIB (fetal dpm/g placenta) was maintained in the ligated horn, feto-placental extraction (feto-placental dpm/PBF) was increased, and placental transfer of the substrate analogs (fetal dpm/g fetus) was unchanged. At d 50 and 63, placental wt and FW were reduced in proportion (approximately 40%) in the growth-retarded group and FW/PBF was increased by 80 and 51%, respectively. Placental transfer of AIB was reduced by 33% at d 50 and by 18% at d 63. In addition, placental efficiency to transfer AIB was reduced by 36% at d 50 and by 22% at d 63 in the growth-retarded group. Fetal uptake of MG per g fetus was slightly reduced (-6%) at d 50 of gestation, but unaffected at d 63. Extraction of MG from the maternal PBF was increased, whereas the wt-specific transfer capacity of the placenta was unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of indomethacin on materno-fetal amino acid transfer in the dual-perfused human placental cotyledon

Indomethacin (1, 10 and 100 mumol L-1) was incrementally infused (8 min for each concentration) into the maternal and fetal circulations of dual-perfused human placental lobules, and measurements were made of materno-fetal amino acid transfer (using the non-metabolizable amino acid alpha-aminoisobutyric acid, or AIB) and fetal effluent perfusate prostaglandin (PG) E levels. No significant changes in AIB transfer compared with controls were observed at any of the doses of indomethacin infused. There was, however, a significant dose-dependent reduction compared with controls in PGE in the fetal circulation with the infusion of indomethacin.

Placental blood flow and transfer of nutrient analogues during normal gestation in the guinea pig

Simultaneous measurements of placental transfer of 14C-alpha-aminoisobutyric acid (AIB), 3H-methyl glucose (MG) and placental blood flow were performed in anaesthetized guinea pigs at various gestational ages. The results were correlated with the growth of the conceptus. The placenta increased its weight at a maximal rate (g/day) between days 31 and 40 of gestation while maximal fetal increments in weight occurred between days 51 and 60. Total net transfer of AIB and MG per gram of placenta increased progressively during pregnancy while net transfer per gram of fetus decreased approaching term. Placental blood flow in the maternal side increased linearly near term. The quantities of AIB and MG transferred by the placenta per ml of blood was maximal between 51 and 60 days of gestation. These data suggest that a relatively smaller placenta in relationship with fetal size limits the quantity of nutrients transferred to the fetus near term, and reduces the rate of fetal growth.

Effect of Maternal Dietary Restriction on Fetal Growth and Placental Transfer of α-Amino Isobutyric Acid in Rats

Maternal net weight gain, plasma clearance, and placental and fetal accumulation of I.V. administered 14C-α amino isobutyric acid (AIB) on day 20 of gestation were measured in pregnant rats: 1) fed ad libitum throughout gestation (Control), 2) fed 50% of the normal daily food intake during the last week of gestation (catabolic phase), and 3) fed 50% of the normal daily food intake throughout gestation. Dietary restriction during the catabolic phase only resulted in a loss of 54% of the net maternal weight gained during the first two weeks of gestation, while dietary restriction throughout gestation resulted in a net loss of 5.4% of the dams prepregnant weight. Both dietary regimens caused significant growth retardation of the fetus. Maternal plasma clearance of AIB was rapid and occurred at the same rate in all three groups. Placental accumulation and transfer of AIB to the fetus was reduced relative to the controls only in dams fed the restricted diet throughout gestation. Fetal to placental AIB concentration ratios were similar for each group indicating no difference in ability of placentas to release amino acid into the fetal circulation. The results indicate that mechanisms other than reduced nutrient transfer may be responsible for fetal growth retardation induced by maternal malnutrition during the catabolic phase of pregnancy only.dietary restriction pregnancy fetal growth placental transfer

Placental Blood Flow and Transfer of Nutrient Analogs in Large, Average, and Small Guinea Pig Littermates

Placental blood flow in the maternal side and transfer of [14C]-alpha isobutyric acid (AIB) and [3H]methylglucose (MG) were measured in 22 pregnant guinea pigs at various gestational ages. The fetuses were divided in three groups according to their body weights: small, average, and large. Body weight was 85.25% of average values in the small fetuses and 114.12% in the large fetuses. Placental weight was 121.73% of average in the large fetuses and 84.42% in the small fetuses. Placental blood flow was 134.48% of average in large fetuses and 73.18% in small fetuses. AIB and MG transfer were significantly lower in the small fetuses (80.33% and 86.06%, respectively, of average values). In contrast, in large fetuses, AIB transfer was 123.43%, and MG transfer was 113.96% of average. Significant correlations were found between fetal and placental weight and placental blood flow and transfer of AIB and MG in the various groups. Placental transfer of AIB and placental blood flow were significantly correlated in the small (r = 0.59) and average weight fetuses (r = 0.37). In addition, the slope of the regression curve for AIB was significantly steeper in the small fetuses when compared with the slope of average and large fetuses. Placental blood flow and transfer of MG were significantly correlated only in the large fetuses (r = 0.48). In the small fetuses, the rate of AIB transfer was proportionally more reduced than that of MG transfer as the rate of placental blood flow decreased. The results demonstrate that spontaneous fetal growth retardation in the guinea pig is associated with a smaller placenta, a reduced placental blood flow, and a reduced transfer of AIB and MG. Inasmuch as in the small fetuses AIB transfer was proportionally more reduced than MG transfer, it is suggested that in addition to the reduced blood flow limited availability of certain essential amino acids may be a cause of fetal growth retardation.

Placental amino acid uptake: V. Relationship to placental maturation in the rat

The functional maturation of the placenta during the latter portion of pregnancy is almost certainly essential to fetal growth but its mechanism is largely unknown. To determine the role of changes in intrinsic cellular transport in this process wer measured the activity of transport systems for AIB between day 14 and term, a period of known marked increase in in vivo AIB transfer in the rat. In vitro incubation demonstrated that the labyrinthine tissue possessed two transport systems for cellular AIB uptake. Their maximum velocities remained essentially constant from day 16 to term and the intracellular concentration achieved during incubation actually decreased with gestational age. In vitro tissue preincubation increased cellular uptake of AIB and this response also decreased with maturation. Thus changes in intrinsic transport mechanisms do not at all parallel the very large maturational increase in in utero amino acid transfer. Changes in intrauterine factors such as blood flow, the hormonal millieu, or fetal utilization and the resultant placental-fetal concentration gradients are much more likely to account for the increase in transfer that are alterations in cellular transport mechanisms.

210 PLACENTAL BLOOD FLOW AND TRANSFER OF NUTRIENTS IN SPONTANEOUS FETAL GROWTH RETARDATION IN THE GUINEA PIG

Reduced availability of nutrients due to reduced placental blood flow or placental insufficiency is considered the most likely cause of intrauterine growth retardation(IUGR). These two variables, however, have not been measured during spontaneous IUGR. Since the guinea pig has a high incidence of spontaneous IUGR, it was, therefore, selected as a suitable model to explore the mechanisms of IUGR. Simultaneous measurements of maternal placental blood flow, using microspheres, and placental transfer of 14C-α-amino isobutyric acid(AIB) and 3H-3-0-methyl glucose(MG) were made between 32 and 59 days of normal gestation. Small (S) and large(L) littermates were compared to mean litter values. Results are as follows:fetal weight:S-18%,L+12%; placental weight:S-16%,L+14%; blood flow: S-35%,L+27%; total AIB transfer:S-34%,L+29%; total MG transfer:S-18%,L+13%;AIB transfer/g placenta:S-20%, L+16%; AIB transfer/g placenta/ml blood flow(placental efficiency):S+17%,L-13%. Thus in the guinea pig fetal growth is correlated with placental blood flow. IUGR fetuses have a reduced blood flow without a concomitant reduction in the efficiency of the placenta to transfer nutrients. (Supported in part by USPHS, NIH Grant KO4 HD 00116-01 & by the R.S. McLaughlin Found.)

MATERNO-FETAL TRANSFER AND UPTAKE OF 2-DEOXY-GLUCOSE (DG) AND α-AMINOISOBUTYRIC ACID (AIB) IN INTRAUTERINE GROWTH RETARDATION (IUGR) ASSOCIATED WITH RESTRICTED UTERINE BLOOD SUPPLY

The mechanism by which restriction of uterine blood flow causes IUGR is not clearly established. We induced IUGR in rat fetuses by ligation at 18 days gestation of the artery supplying one uterine horn. Two days later, the pregnant rats received intravenously 25 μC H3-DG (10 C/mM) and 1 μC C14-AIB (9 mC/mM) per 100 gm. After 60 minutes IUGR and control (unligated horn) fetuses and placentas were delivered by C-section, and radioactivity determined (dual channel) in homogenates of placenta, fetus, liver, and brain, and in fetal and maternal plasma. Plasma concentrations and fetal/maternal ratios of both AIB and DG averaged 35% less in IUGR. Uptake of AIB per gram tissue by placenta, whole fetus, brain, and liver was 34, 44, 37 and 22% smaller respectively in IUGR. Uptake of DG per gram tissue by placenta, whole fetus, and brain was reduced 14, 15, and 16% respectively in IUGR. (All differences, p<.05). In contrast, liver uptake of DG averaged 17% greater in IUGR (p=0.2). The ratio of DG in fetal liver/fetal plasma, and in fetal brain/fetal plasma, was greater in IUGR than control (both p<.05). We conclude that in this IUGR model: there is reduction in transfer of DG and AIB into most fetal tissues; reduced growth in utero can reasonably be explained by diminished supply of amino acids and glucose; the IUGR fetal liver adapts glucose uptake more effectively than does fetal brain to reduced glucose supply.

PLACENTAL TRANSFER OF NUTRIENTS DURING NORMAL AND PROLONGED GESTATION

The rate of placental transfer of glucose and α-amino isobutyric acid (AIB) was studied at 14, 17, 18, 19, 20 and 21 days of gestation in normal rats and at day 22 and 23 (prolonged gestation) in rats with uterine ligation. Transfer of both substances increases linearly from day 14 to day 19, levels off between days 19 and 20 and drops to 75 per cent of day 20 levels at day 21. There is a 90 per cent increase in AIB transfer between days 14 and 20 compared with a 28 per cent increase in glucose transfer during the same period. Thus, although the developmental pattern was similar the capacity of the placenta to transfer amino acids develops later than glucose. Prolonged gestation markedly reduces the capacity to transfer AIB but has little effect on the placenta ability to take up this substance. This was demonstrated in experiments showing little if any change in uptake of AIB at days 22 and 23 compared to day 21. By contrast, AIB transfer was 30 per cent less than day 21 and 55 per cent less than day 20. Thus, there are marked developmental differences in the maturation of the transfer systems for glucose and amino acids. Prolonged gestation reduces transfer of both substances.

MATERNAL MALNUTRITION AND PLACENTAL TRANSFER OF NUTRIENTS IN THE RAT

It is now well established that maternal malnutrition will retard fetal growth. It has been assumed that the primary mechanism producing this growth retardation is through a reduction in nutrient supply. Since maternal malnutrition affects cellular growth of placenta itself, it is possible that placental function as manifested by its ability to transfer nutrients is also impaired. Placental transfer of glucose and α-amino isobutyric acid (AIB) was measured after maternal protein restriction. Glucose transfer into the fetus on days 20 and 21 of gestation was reduced by about 60 per cent. AIB transfer was reduced by 37 per cent on day 20 and 47 per cent on day 21. By contrast maternal plasma glucose levels, the rate of glucose disappearance from the plasma and of glucose incorporation into maternal tissues was not altered by maternal protein restriction. These data demonstrate that placental function as manifested by the ability to transfer both glucose and amino acids is impaired by maternal protein restriction. It is possible that the fetal growth retardation is secondary to this placental “dysfunction.”

Active transport of amino acids into bone cells

The transport of the model amino acid α-aminoisobutyric acid (AIB) into bone cells isolated mechanically from metaphyses of 40-day-old rats has been examined. Net transfer of AIB into bone cells against a concentration gradient was observed. This process was temperature and energy dependent, thus fulfilling the criteria for “active” transport. Intact cell membranes were required. Glucose concentration in the medium, attempts to manipulate the intracellular electrolyte composition by means of leaching and variations of external calcium, magnesium and phosphate had no effect on this system. Two-thirds of the transport of AIB into bone cells was dependent on the concentration of sodium and potassium in the external medium. It is therefore suggested that two different mechanisms are involved, one independent and the other dependent on ambient sodium and potassium concentrations. The fact that the latter shares a group of properties with the cation-dependent ATPase system points to a possible relation between these two mechanisms. The experiments reported here suggested the possibility that this active transport step might represent a site of control in the biosynthesis of collagen and other proteins in this tissue.