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.

Adrenergic innervation of blood vessels in rat tibial nerve during Wallerian degeneration.

Adrenergic innervation of blood vessels in rat tibial nerve during Wallerian degeneration was examined, using the formaldehyde-induced histo-fluorescence method. The left sciatic nerve was transected at the level of the sciatic notch, whereas the right sciatic nerve was left intact and used as control. At 1, 3, 7, 14, 42, 56 or 84 days after transection, the tibial nerves of the transected and contralateral sides were exposed. Pieces of each nerve were used for light microscopy or for examination of adrenergic innervation with the fluorescence microscope. One day after transection, no adrenergic nerve fiber was observed in the endoneurium of the transected nerve. After 3 days, adrenergic innervation of small- and medium-sized arterioles in the epi-perineurium was absent, and after 7 days no fibers were visible around large arterioles. Fluorescent fibers were not detected even at 84 days post-surgery. It is concluded that adrenergic innervation of blood vessels in the rat tibial nerve is irreversibly lost after permanent axotomy, and that adrenergic regulation of nerve blood flow may also be lost.

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 MAP's and CV's 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 MAP's and CV's 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.

Cerebral metabolism of plasma [14C]palmitate in awake, adult rat: subcellular localization.

Following intravenous injection of [U-14C]palmitate in awake adult rats, whole brain radioactivity reached a broad maximum between 15-60 min, then declined rapidly to reach a relatively stable level between 4 hr and 20 hr. At 44 hr total radioactivity was 57% of the 4 hr value (p less than 0.05). About 50% of palmitate which entered the brain from the blood was oxidized rapidly, producing 14C-labeled water-soluble components which later left the cytosol. Radioactivity in the cytosolic fraction peaked at 45 min and then declined, coincident with the decline in total brain radioactivity. Membrane fractions were rapidly labeled to levels which remained relatively stable from 1 to 44 hr. Increases in the relative distributions of radioactivity were seen between 1 and 4 hr for the microsomal and mitochondrial fractions, and beyond 4 hr for the synaptic and myelin membrane fractions (p less than 0.05). Radioactivity in membrane fractions was 80-90% lipid, 5-13% water-soluble components and 3-17% protein. The proportion of label in membrane-associated protein increased with time. Proportions of radioactivity in the combined membrane fractions increased from 65% to 76% to 80% at 4, 20 and 44 hr, respectively. The results show that plasma-derived palmitate enters oxidative and synthetic pathways to an equal extent, immediately after entry into the brain. At and after 4 hr, the radiolabel resides predominantly in stable membrane lipids and protein. Brain radioactivity at 4 hr can be used therefore, to examine incorporation of palmitate into lipids in vivo, in different experimental conditions.

Assessment of the permeability of the blood-retinal barrier in hypertensive rats.

We assessed the permeability surface area products at the blood-retinal barrier and blood-brain barrier to sucrose (molecular weight, 340) and microperoxidase (molecular weight, 2000) following acute hypertension induced by metaraminol in Wistar-Kyoto rats (controls) and during chronic hypertension in spontaneously hypertensive rats. In acute hypertension, the permeability surface area product for sucrose was increased at the blood-retinal barrier and at the blood-brain barrier over control values (p less than 0.02), and the vessels became leaky to microperoxidase. In chronic hypertension, the permeability of the blood-retinal barrier to sucrose was increased over that in control animals (p less than 0.02), whereas the permeability of the blood-brain barrier was unaffected. Neither barrier leaked microperoxidase. These results indicate that the blood-brain barrier and the blood-retinal barrier are similarly affected in acute hypertension and that in chronic hypertension, the blood-brain barrier is unaffected whereas the blood-retinal barrier is rendered more permeable to small, but not large, solutes.

Differential localization of alkaline phosphatase in barrier tissues of the frog and rat nervous systems: a cytochemical and biochemical study.

We investigated the localization of alkaline phosphatase (AP) in the peripheral and central nervous systems of the frog (Rana pipiens) and rat. In the frog sciatic nerve, AP reaction product was seen as a precipitate within caveolae and vesicular profiles of perineurial cells, and frequently filled the extracellular space. In the rat peripheral nerve, AP reaction product appeared as small tufts on the cell surfaces and within vesicular profiles of endoneurial blood vessels. AP reaction product was not detected in the rat perineurium or in endoneurial blood vessels of the frog. In the frog central nervous system, AP reaction product was detected in the arachnoid membrane adjacent to the subarachnoid space, but not in the cerebral or pial vessels, whereas in the rat it was detected in the outer arachnoid membrane and in the cerebral and pial blood vessels. Biochemical analysis indicated a sevenfold higher AP activity in the frog perineurium over the endoneurium, whereas in the rat, threefold more activity was measured in the endoneurium over the perineurium. Levamisole, an AP inhibitor, decreased the enzyme activity by 95% in rat tissues, and by 70% in frog tissues and in plasma from both animals. Similar decrements were observed cytochemically. This study suggests that: (1) the distribution of AP varies between species, but that it is always present in at least one component of the blood-brain and blood-nerve barriers, (2) because barrier tissues of the nervous system have enzymatic activity, they may biochemically modify the adjacent environment, (3) vesicular profiles and caveolae in the blood vessels and perineurium may function as microenvironments for enzymatic activity, and (4) in the rat and frog, different isozymes of AP may be present.

Altered blood-nerve barrier permeability to small molecules in experimental diabetes mellitus.

Permeability-surface area products (PA) were determined with a quantitative in vivo injection technique at the blood-nerve barrier of tibial nerve, and at the blood-brain barrier, in control and streptozotocin-induced diabetic rats. The PA product for [14C]mannitol at the blood-nerve barrier was increased by 100% in diabetic animals, 3.12 +/- 0.15 X 10(-5) ml X s-1 X g-1, compared with controls, 1.61 +/- 0.10 X 10(-5) ml X s-1 X g-1. In contrast, PA for [14C]mannitol at the blood-brain barrier was unaltered in the diabetic animals. Following intravenous injection, no leakage of microperoxidase across the perineurium or endoneurial vessels of diabetic rats could be demonstrated by morphological techniques. Nerve blood-space, as determined with intravenous [3H]inulin, and blood-nerve barrier surface area as determined by morphometric methods, did not differ in diabetic when compared to control animals. Thus, the calculated permeability coefficient for [14C]mannitol at the blood-nerve barrier was about 100% greater in diabetic nerve compared to control nerves. The increased permeability was accompanied by a 7% increase in nerve-water content and a 32% decrease in motor-nerve conduction velocity. The results demonstrate a specific vulnerability of nerve as compared to brain in an animal model of diabetes mellitus. Chronically altered permeability to small water-soluble molecules reduces the protective effect of salt impermeability at the blood-nerve barrier against nerve edema, and may be an important pathogenic mechanism in diabetic neuropathy.

Regression of blood vessels precedes cartilage differentiation during chick limb development.

We have previously investigated distinct areas of vascular regression in the developing vascular system of the chick limb bud. Avascular areas appear in a characteristic spatial and temporal pattern, and are correlated with the position of developing cartilage. In the present study, we examined limb-bud sections which had been double labeled for endothelial cells and developing cartilage in order to determine the relationship between the appearance of cartilage and the disappearance of capillaries. Endothelial cells, which specifically take up acetylated low-density lipoprotein (acLDL), were labeled by intravenously injecting fluorescent acLDL (DiIacLDL) into chick embryos at Hamburger and Hamilton stages 26-30. Avascular zones, which correspond to the developing digits, were clearly visible within the fluorescently labeled distal vasculature. The same sections were labeled with monoclonal antibodies specific for cartilage. We found that progressing avascularity in the digital regions was followed by increased staining for cartilage antigens in the same areas. Zones of avascularity always developed earlier than morphologically and immunologically detectable cartilage in all planes of section and were always larger than the areas of cartilage. These results demonstrate that blood vessels disappear in predictable areas prior to the overt differentiation of cartilage.

The morphology of pial blood vessels of the frog, preserved by rapid freezing and freeze substitution.

Vesicular profiles in endothelial cells of frog meninges were examined in tissues preserved by rapid freezing or conventional chemical fixation. Tissues were frozen immediately after removal from the animal or after remaining in Ringers for several hours. Vesicular profiles with an average diameter of 240 nm were present in the endothelial cells in all experimental groups, demonstrating that they are not an artifact of chemical fixation or incubation in vitro. However, their concentration and morphology varied with the different preservation techniques.

Localized vascular regression during limb morphogenesis in the chicken embryo. I. Spatial and temporal changes in the vascular pattern.

Morphogenesis of the vertebrate limb bud depends upon reciprocal interactions between epithelial and mesenchymal tissues. A characteristic limb vascular pattern is essential for normal limb outgrowth. The vascular pattern in the distal portion of the wing bud was examined by ink injection and compared to the sites of cartilage differentiation, as indicated by [35S]-incorporation. During development, avascular areas arose in three distinct locations within the vascularized mesoderm. These areas corresponded to the distal skeletal elements, referred to as digits 2, 3, and 4. Incorporation of radioactive sulfate was high in the avascular areas and low in the adjacent vascular tissue. Examination of autoradiographs of ink-injected limbs suggested that the appearance of avascular regions preceded the accumulation of sulfated cartilage matrix. These results indicate that remodeling of the limb vasculature is related to the formation of the skeletal pattern.

Localized vascular regression during limb morphogenesis in the chicken embryo: II. Morphological changes in the vasculature.

The regression of blood vessels in the distal wing bud of chicken embryos from stages 19 to 31 was examined by light and electron microscopy. The vessels were double-labelled by an injection of Monastral blue B (MB) to label the regressing endothelial cells, followed 6-48 hours later with an injection of India ink which marked the lumens of patent vessels. Prior to stage 26 the vessels contained only India ink since the endothelial cells were not phagocytic at this stage. Vessels at stage 26 or later were often double-labelled, with MB sequestered in the endothelial cell cytoplasm and India ink in the vessel lumens. After stage 27 cells not associated with lumens, but labelled with MB, were observed in areas undergoing vascular regression. Ultrastructural changes in the endothelial cells as the vessels regressed included formation of luminal and abluminal processes, long complex junctions, and vacuoles containing MB. In many involuting vessels the endothelial cells appeared normal even though the lumens were collapsed. Occasionally, isolated pyknotic cells were observed in regions that had been previously vascularized. At stage 31 cells in the developing cartilage had vacuoles containing MB. Our study suggests that blood vessels may disappear from the prechondrogenic zone of the distal wing bud by several mechanisms. These could include a type of cell death that does not elicit a cellular infiltrate, migration of the endothelial cells away from vascularized regions, and/or transdifferentiation into cells that resembled chondrocytes.

Transport and steady-state concentration of plasma proteins in the vitreous humor of the chicken embryo: implications for the mechanism of eye growth during early development.

The nature and origin of the proteins of the vitreous humor were examined in chickens during embryonic and early posthatching stages. The major proteins of the vitreous humor were similar in electrophoretic mobility to plasma proteins at all ages examined. Earlier studies from our laboratory and experiments described below showed that plasma proteins continuously entered and left the eye throughout its development. From these data it was concluded that the majority of vitreous-humor proteins were derived from the blood. The protein concentration of the vitreous humor was 13% of that of the plasma from embryonic Days 6 through 15 (E6 through E15). After E15, the relative protein concentration in the vitreous humor declined with respect to the plasma and reached 4% of the plasma protein concentration at hatching. Several possibilities were considered to account for how proteins can rapidly enter and leave the eye, yet maintain a steady-state concentration in the vitreous humor that is less than one-seventh of that in the blood.

Vesicular profiles in frog perineural cells preserved by rapid-freezing and freeze-substitution.

Vesicular profiles in perineurial cells of frog peripheral nerves were examined in tissues preserved by rapid-freezing or by conventional chemical fixation. Tissues were processed immediately after removal from the animal or after remaining in Ringers for several hours. Vesicular profiles were present in perineurial cells in all experimental groups, demonstrating that they are not an artifact of chemical fixation. However, variations in their morphology correlated with the different preservation techniques.

Developmental changes in the blood-ocular barriers in chicken embryos.

Changes in the permeability characteristics of the developing chicken eye were studied using light and electron microscopy. Chicken embryos from 6-19 days of gestation (E9-E19) and within 1 day of hatching (P1) were injected intravascularly with horseradish peroxidase (HRP) and examined 1 and 5 min after injection. Within 1 min of injection there was focal accumulation of horseradish peroxidase reaction product (HRP-RP) at the angle of the anterior chamber in embryos up to E15. By 5 min post-injection the HRP diffused into the anterior and posterior chambers and vitreous body; and extended posteriorly into the developing uveal tract. No leakage was detected in the E19 or older animals. In the posterior segment of the eye prior to E12, HRP from the adjacent connective tissues diffused into zone III of the optic nerve. After E12 the developing meninges prevented the influx of the HRP. At the level of the lamina cribosa HRP permeated zone II up to E19 after which only 25% of the animals examined showed HRP-RP in this area. In zone I in all ages examined no HRP-RP was detected 5 min after injection. Developing blood vessels in the deep iris stroma, optic nerve and pecten remained impermeable even as they grew, while the choroidal vessels were consistently leaky. This study suggests: (1) that proteins from the vascular system reach the intraocular chambers before E19; (2) that the leakage occurs from vessels located in the uveal tract adjacent to the angle; (3) that the permeability of the optic nerve depends on the development of the meninges and the border tissues associated with the lamina cribosa; and (4) that the growing blood vessels in the developing eye have permeability characteristics similar to those found in mature vessels.

Seeding of Dacron vascular prostheses with endothelium of aortic origin.

Seeding of autologous venous endothelium on Dacron vascular prostheses in dogs results in endothelial coverage of the prosthetic flow surface 4-6 weeks after implantation. Canine aortic endothelium, in contrast, usually fails to completely cover an unseeded prosthesis by pannus ingrowth even over much longer periods. To see if the success of endothelial seeding stems from a difference in the ability of venous and aortic endothelium to grow on prosthetic surfaces, we seeded freshly harvested autologous aortic endothelium on Dacron velour infrarenal aortic prostheses in dogs. Six weeks after surgery these prostheses showed the features reported to be typical of seeded prostheses. Scanning electron micrographs showed a luminal lining of flat polygonal cells without fibrin or adherent formed blood elements, and light microscopy showed an underlying layer containing aligned spindle-shaped cells with elongated nuclei and cell-lined subluminal channels. Control prostheses were covered with fibrin and platelet-rich thrombi everywhere except for limited pannus ingrowth at anastomotic sites. The results suggest that the success of autologous endothelial seeding cannot be ascribed to inherent differences in properties such as mitotic capacity or fibrinolysis between venous and aortic endothelium. The formation of complete endothelial linings by seeding must instead result from a more favorable condition for endothelial cell growth created by the cell harvesting or seeding process itself.

Early normalization of platelet survival by endothelial seeding of Dacron arterial prostheses in dogs.

Current synthetic vascular prostheses do not acquire a complete lining of vascular endothelium in humans or dogs. Seeding of autogenous venous endothelium has bee reported to remedy this defect, but previous studies have been primarily morphologic. To see if venous endothelial seeding caused a measurable decrease in platelet-prosthetic interaction in vivo, serial 111In-labeled platelet survival studies were done in dogs with seeded (N = 8) and unseeded (n = 9) thoracoabdominal Dacron bypass prostheses.l Changes in platelet survival time (PST) were compared with endothelial coverage scores, which were determined by blinded survey of removed prostheses by scanning electron microscopy (SEM). The PSTs before surgery (+/- SD) were 5.02 +/- 0.79 days (seeded) and 5.31 +/- 0.99 days (control) (P = 0.51). Seven weeks after surgery the PSTs were 4.01 +/- 1.10 days (seeded) and 2.67 +/- 0.88 days (control) (P = 0.013). The subgroup of four dogs with seeded prostheses that had complete endothelial coverage showed restoration of the platelet survival pattern to that of the linear decay seen before surgery, SEM studies showed that this normalization of PST occurred despite the presence of small nidi of platelet adhesion on exposed Dacron fibers and irregularities of the endothelial pattern consistent with flow disturbances and venous jet lesions near anastomoses. Endothelial seeding, when technically successful, appears to sharply curtail the degree of platelet interaction with vascular prostheses and restores a normal platelet survival pattern. Longer studies of seeded prostheses are needed to determine if seeding protects against the late complications of intimal hyperplasia and thrombotic occlusion.

Regression of the tunica vasculosa lentis in the postnatal rat.

Involution of the tunica vasculosa lentis (TVL) was examined in albino rats from 1 to 21 days after birth. Scanning electron microscopy demonstrated that as the lens increased in size, the larger vessels became straight and the small interconnecting vessels disappeared. Transmission electron microscopy revealed that the endothelial cells lost their close relationship with the posterior lens capsule. In the endothelial cells and pericytes the cytoplasm was dense, and many of the organelles became to recognize. However, the cells appeared to remain intact for into regression, shrinking until a basement membrane-like remnant remained. Vitreal cells were close to the TVL throughout the period studied, but few contained phagosomes. During regression the TVL appeared to (1) change its shape in order to accommodate the enlarging ocular structure, (2) maintain separation of the vitreal and vascular compartments by remaining intact during regression and (3) be associated with vitreal cells, which did not seem to play a prominent role in the involution of these vessels.