Armin Ermisch

Chapter 27: Age-related pathophysiology of the blood-brain barrier in heat stress

The possibility that the blood-brain barrier (BBB) might play an important role in the pathophysiology of heat stress (HS) has been examined in young (age 8-9 weeks) and adult (age 24-32 weeks) rats. Exposure of young rats to 4 h HS at 38 degrees C in a biological oxygen demand (BOD) incubator (relative humidity 47-50%, wind velocity 20-26 cm/sec, simulating the environmental conditions of Varanasi, India, during the month of June) resulted in a marked hyperthermia (41.7 +/- 0.23 degrees C) and behavioral symptoms. In these animals there was a profound increase in the permeability of the BBB to Evans blue-albumin (EBA) (464%) and to 131I-sodium iodide (515%), accompanied by a marked increase in the brain water content (4%), of the levels of serotonin (5-hydroxytryptamine, 5-HT) in plasma (687%) and in brain (267%) and a pronounced reduction (30%) in cerebral blood flow (CBF). Morphological examination using light- and electron-microscopy revealed profound neuronal changes associated with a marked increase in glial fibrillary acidic protein (GFAP) and in vimentin immunoreactivities, together with a substantial reduction in myelin basic protein (MBP) immunostaining in the brain. These changes were more pronounced in the brain-stem reticular formation, pons and medulla region. On the other hand, exposure of adult animals to the same intensity of HS resulted in mild or no changes in BBB permeability, content of brain water and 5-HT in the plasma and brain, CBF or other cellular changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin binds to microvessels from rat hippocampus.

Recent evidence suggests that vasopressin may influence the permeability of the endothelium of brain capillaries. We measured the binding of [125I]arginine-8-vasopressin ([125I]AVP) to microvessels isolated from different regions of the rat brain. The study revealed saturable and specific binding of [125I]AVP to microvessels isolated from hippocampus. Scatchard analysis confirmed a single class of high affinity sites with an equilibrium dissociation constant, Kd, of 3.2 nM and an apparent maximal binding capacity of 205 fmol/mg protein. No binding was observed to microvessels from neocortex and striatum.

On the blood-brain barrier to peptides : specific binding of atrial natriuretic peptide in vivo and in vitro

Using the intracarotid bolus injection technique, a saturable binding of [125I]atrial natriuretic peptide (ANP) was found in 8 blood-brain barrier (BBB)-protected rat brain regions as well as in the pineal gland, choroid plexus, neurointermediate and anterior lobes of the pituitary, i.e. structures lacking a BBB. The presence of specific ANP binding on the BBB, here shown for the first time by an in vivo approach, was evidenced concomitantly in vitro by incubation of isolated microvessels. A single-class high affinity binding without regional differences was obtained with Kd = 0.23 nM and Bmax = 120 fmol/mg protein. From that a density of 1,400 binding sites per endothelial cell was calculated, thought to be localized predominantly in the luminal membranes. In the in vivo study, the portion of the extracted peptide that, under the conditions used, may have crossed the BBB by passive diffusion amounted to less than 0.4% of the labeled ANP administered. ANP itself did not change the tightness of the BBB to the non-diffusible reference molecule [14C]inulin. In the BBB-free areas, ANP enhanced the inulin space by nearly 50%.

Saturable Retention of Vasopressin by Hippocampus Vessels In Vivo, Associated with Inhibition of Blood-Brain Transfer of Large Neutral Amino Acids

Abstract: Vasopressin receptors have been reported in the endothelium of brain capillaries. The function of these receptors is not known. To test the prediction that vasopressin receptors in brain capillary endothelium affect amino acid transport across the blood‐brain barrier and to assess the role of vasopressin transport across the cerebral vascular endothelium, we measured (a) the endothelial permeability to the large neutral amino acid leucine in the absence and presence of arginine vasopressin (AVP) and (b) the permeability of the blood‐brain barrier to AVP relative to mannitol. In brain regions protected by the blood‐brain barrier, after circulation for 20 s, coinjection of leucine and AVP intravenously led to a decrease of leucine transport unrelated to changes of blood flow. The decrease was most pronounced in hippocampus (42%) and least pronounced in olfactory bulb and colliculi (17 and 19%, respectively). In the latter regions, the endothelial permeability to AVP did not significantly exceed that of mannitol. In hippocampus and in regions with no blood‐brain barrier (pituitary and pineal glands), AVP retention in excess of mannitol retention was blocked by unlabeled AVP. The findings do not contradict the hypothesis of a role for AVP in the regulation of large neutral amino acid transfer into brain tissue.

Simple high-performance liquid chromatographic analysis of free primary amino acid concentrations in rat plasma and cisternal cerebrospinal fluid.

The quantitation of 16 acidic, basic, small and large neutral amino acids was performed using 10-microliters sample aliquots of cisternal cerebrospinal fluid (CSF) and blood plasma of rats. The analytical technique is based upon a two-buffer HPLC system with fluorimetric detection of pre-column derivatized primary amino acids with o-phthaldialdehyde (OPA). A modification of a well established method, the power of the present technique comes from an improved resolution and sensitivity by installing a column heater adjusted to 43 degrees C and strictly reducing any contamination by background amino acids. The analysis is simplified by separating the amino acid derivatives with a linear buffer gradient and less time consuming by the use of a short analytical column with a higher flow-rate. Analytical precision, linearity of response and reproducibility were highly acceptable at both CSF and plasma concentrations of amino acids without changing any of the separation or detection parameters.

Arginine vasopressin reduces the blood-brain transfer of l-tyrosine and l-valine: further evidence of the effect of the peptide on the l-system transporter at the blood-brain barrier

Arginine vasopressin (AVP) coinjected into the carotid artery in physiological concentrations (0.1 nmol/l), with either L-[3H]tyrosine or L-[3H]valine, induced changes in the kinetic parameters of the blood-to-brain transfer of both large neutral amino acids (LNAA) without alterations in brain haemodynamics. The half-saturation constant (Km), the maximum velocity of transport (V(max)) and Kd, the nonsaturable transport constant, were estimated in 9 brain regions of male Wistar rats anaesthetized with ether. Apart from Kd, significant changes in Km and V(max) were observed in all brain regions investigated. On average Km decreased from 0.17 to 0.048 mmol/l for tyrosine, and from 0.61 to 0.059 mmol/l for valine, whereas V(max) declined from 22 to 9.9 nmol/min/g for tyrosine, and from 29 to 3.2 nmol/min/g for valine, respectively. The results provide further evidence that vasopressin-receptor interactions at the blood-brain barrier (BBB) induce changes in the properties of the common transporter, the L-system, which eventually result in a suppression of the blood-to-brain transfer of LNAA. Data analysis of the 5 LNAA tested so far reveals a significant negative correlation (R = 0.98, P < 0.05) between the respective substrate affinity for the transporter and the corresponding magnitude of transport reduction induced by circulating AVP. Calculations of the unidirectional influx (J) of the LNAA indicate that AVP (1) reduces J by approximately one-third for every LNAA, but (2) does not change the relative contribution for each single LNAA to the total influx across the BBB.

Changes in amino acid levels in rat plasma, cisternal cerebrospinal fluid, and brain tissue induced by intravenously infused arginine-vasopressin

Circulating arginine-vasopressin (AVP) is known to reduce the blood-to-brain transfer of large neutral amino acids (AA). As a first step to examine whether the reduced uptake by brain endothelial cells is reflected in changes in large neutral amino acid levels of the extracellular fluid environment of cells within the nervous tissue, we measured the concentrations of amino acids in plasma, cerebrospinal fluid (CSF), and hippocampal tissue of rats before and after infusion of AVP (34 and 68 ng/min/kg, respectively) over the time period of 60 min. AA levels changed in all compartments investigated during both saline and AVP infusions. Whereas in the saline-infused controls changes in CSF AA levels paralleled those in plasma, this correlation was abolished by raising AVP concentrations. The effect of AVP was found to be i) dependent on the AA, ii) different with respect to direction and iii) magnitude of changes in AA levels, and iv) in some cases dose dependent. In summary, AVP infusion increased plasma levels of 10 AA, but decreased all 15 AA measured by some 30% in CSF. In contrast to CSF, levels of AA were slightly enhanced in the hippocampal tissue. The results are not solely explicable by a reduced blood-to-brain transfer of AA. We conclude that further mechanisms by which AVP affects the availability of AA to the brain may exist. The physiological significance of the findings might be related to brain osmoregulation, especially in situations of stress.

Autoradiographische Untersuchungen über das Wachstum des Reissnerschen Fadens von Cyprinus carpio (L.)

SummaryAutoradiographs of the subcommissural organ (SCO), the Reissners fibre (RF), the ependymal horder and other areas of the brain, as well as non-neural organs of the body of the carp, Cyprinus carpio L., were prepared 4 hours to 76 days after the injection of 35S-cystine.1.Four hours after the injection of 35S-cystine labeled secretory material was found in the cisterns of the SCO-cells. The 35S concentration in the SCO and in the RF is generally higher than in all other areas of the brain investigated. As to the quantities of 35S incorporated the tissues investigated range as follows: Islets of Langerhans>RF>plexus>neurosecretory cells>ependymal border>cerebellum>commissura posterior.2.The daily growth rate of RF is 0.5–2.3% of total length of the cerebrospinal canal from SCO to the filum terminale. The substances making up the RF may pass the third and fourth ventricles and the central canal of the spinal cord within 42–221 (average 64) days. The quantity of secretion released by the SCO of the carp into the third ventricle amounts to 0.3×10−4 mg—4.0×10−4mg daily (rate of secretion). That amounts to 0.5–5% of the total weight of the organ.3.The entire RF from the SCO to the terminal ampoule of the central canal weighs 1.7x 10−2mg. This is up to 260 mg of RF material per 100 g of cerebrospinal fluid. During its growth RF releases no labeled substances. Labeled material gets into RF from the surrounding cerebrospinal fluid by diffusion.4.35S activity in the ependymal border of the filum terminale and the terminal ampoule, as well as the cerebrospinal fluid and the (non-labeled) RF of these areas is higher than in ependymal border, the cerebrospinal fluid, and the (non-labeled) RF of other spinal cord areas.ZusammenfassungVom Subkommissuralorgan (SKO) und dem Reissnerschen Faden (RF), dem Ependym und anderen Bereichen des ZNS sowie Organen der Leibeshöhle des Karpfens (Cyprinus carpio L.) wurden 4 Std bis 76 Tage nach 35S-Cystinapplikation und histologischer Aufbereitung Autoradiogramme hergestellt und untersucht.1.Markiertes Material in den Sekretzisternen der Zellen des SKO liegt 4 Std nach der Injektion von 35S-Cystin vor. Die 35S-Cystinkonzentration im SKO und im RF ist im allgemeinen größer als die Anreicherung von aktivem Material in den übrigen untersuchten Hirnbereichen. Die Gewebe bzw. Organe können nach der Menge des eingebauten 35S zu folgender Reihe geordnet werden: Langerhanssche Inseln>Plexus>neurosekretorische Zellen>Ependym> Cerebellum>Commissura posterior.2.Das tägliche Wachstum (Wachstumsrate) des RF beträgt 0,5–2,3% der Länge des Hirn-Rückenmarkkanales vom SKO bis zur Ampulla terminalis. Die Fadensubstanz passiert das Hirnhohlraumsystem in etwa 42–221 (durchschnittlich 64) Tagen. Die vom SKO des Karpfens abgegebene Sekretmenge (Sekretionsrate) beträgt 0,3·10−4 bis 4,0·10−4 mg/Tag, d.h. etwa 0,5–5% des Eigengewichtes des Organes.3.Die Gesamtmasse des RF vom SKO bis zur Ampulla terminalis beträgt bis zu etwa 1,7·10−2 mg. Dieser Wert entspricht einer Menge von etwa 260 mg RF-Substanz auf 100g Liquor. Der RF gibt während seines Wachstums durch das Hirnkammersystem keine markierten Substanzen ab. Durch Diffusion gelangt Aktivität aus dem umgebenden Liquor in den RF.4.Die 35S-Aktivität in den Ependymzellen des Filum terminale und der Endampulle, im Liquor und im nicht markierten RF dieser Bereiche ist größer als in den Ependymzellen, im Liquor und im nicht markierten RF anderer Rückenmarksabschnitte.

Chapter 23: Peptide receptors of the blood-brain barrier and substrate transport into the brain

The BBB is a target for some peptide signals, as demonstrated by our group for arginine-vasopressin (AVP) and atriopeptin (ANP). Peptide molecules contacting the luminal surface of endothelial cells interact with specific high-affinity binding sites. The minimal simple diffusion of peptide molecules across the layer of endothelial cells which are connected by tight junctions is most probably without any significance under physiological conditions, although that question should be checked for brain regions like the olfactory bulb in which some leakiness of the BBB can be demonstrated. The AVP- and ANP-receptors at least partly localized at the luminal surface of the endothelial cells are heterogeneously distributed in the vessels of the brain. The number of AVP receptors is up-regulated by ligand deficiency, which induces furthermore a decrease in the receptor affinity. At physiological concentrations AVP and ANP do not affect the tightness of the BBB, but regulate the transcellular transfer of essential substances from blood to brain. AVP decreases the Km and Vmax of the transporter of large neutral amino acids, and ANP alters the water permeability of the endothelial cell layer. The phenomenon that the cells of the tight epithelium representing the BBB need information from blood-borne peptide signals for the regulation of intercompartmental transport processes seems to be only a special case of a general principle concerning tight epithelial cell layers which separate compartments containing fluids of different composition; amino acid transport across the intestine is regulated by specific peptides contacting that barrier, the casomorphins.

Atrial natriuretic peptide augments the blood-brain transfer of water but not leucine and glucose.

Recent evidence predicts an effect of atrial natriuretic peptide (ANP) on the blood-brain transfer of water. To test this prediction, we measured the blood-brain transfer of water, L-leucine, and D-glucose in 9 brain regions of male rats after intravenous injection of 10 pmol ANP. The peptide elicited an increase of the permeability-surface area (PaS) product of labeled water by 28-108% while the PaS products of leucine and glucose remained unchanged. Cerebral blood flow increased 15-48% while cardiac output and plasma volume in brain did not alter, indicating no change of capillary surface area (CSA). Regionally, the CSA varied from 63 cm2/g (striatum) to 97 cm2/g (colliculi) and the fraction of capillaries contributing to the total vascular volume varied from 29% (olfactory bulb/lobe) to 62% (striatum). The blood-brain barrier (BBB) permeability to water (5.7 micron/s) was an order of magnitude higher than to glucose (0.4 micron/s) or to leucine (0.3 micron/s).