Administration Of Horseradish Peroxidase Research Articles

Layerwise Organization of Neurons Providing Interhemisphere Connections in the Visual Cortex of Cats with Impaired Binocular Vision

The distribution of callosal neurons in the cortical layers was studied in intact (n = 7) and experimental cats with strabismus (n = 10) or monocular deprivation (n = 5) after microiontophoretic administration of horseradish peroxidase into the eye dominance columns of fields 17 and 18 and the fields 17/18 transitional zone. Callosal neurons in cats with impairments to binocular vision were located more deeply in layers II/III and more shallowly in layer IV than in intact cats. In addition, the proportion of callosal neurons in layer IV increased and the proportion in layer II/III decreased in cats with impaired binocular vision, as compared with intact cats, and these changes were more marked in monocular deprivation. These data point to the important role of sensory information in the formation of the laminar distribution of callosal neurons.

Retinotopic Organization of the Posteromedial Area of the Lateral Suprasylvian Sulcus Shown by Analysis of the Pattern of Corticocortical Connections with Field 17 in Cats

Analysis of the structure of intercortical connections was used to study the retinotopic organization of the visual area located in the posteromedial wall of the lateral suprasylvian sulcus (PMLS) in cats. The retrograde axonal tracer horseradish peroxidase (HRP) was administered into the PMLS and initial neurons were studied in field 17. The pattern of the distribution of labeled cells in field 17 after administration of HRP into the projection of the center of the visual field corresponded to the retinotopic map reported in [8]. However, after administration of HRP into the projection of the upper visual field [8], initial neurons in field 17 were found in the representation of the lower periphery of the visual field: from –10° to –60° on the vertical meridian and from 40° to 80° on the horizontal meridian. This non-correspondence in the representations of the upper and lower visual fields has also been seen in electrophysiological and topographical studies [5]; these data provide evidence for the need to use the Grant and Shipp retinotopic map in morphofunctional studies of the PMLS in cats.

Hypoxia-Induced Cellular and Vascular Changes in the Nucleus Tractus Solitarius and Ventrolateral Medulla

Major changes in arterial pressure, autonomic, and respiratory activity occur in response to hypoxia. We analyzed structural damage and increased vascular permeability in the ventrolateral medulla and nucleus tractus solitarius, which control autonomic, respiratory, and cardiovascular functions in adult Wistar rats subjected to 2 hours of hypoxia (7% oxygen + 93% nitrogen) for up to 14 days after hypoxicexposure. Brainstem tissue levels of vascular endothelial growth factor (VEGF), nitric oxide (NO), and glutamate were significantly increased over control levels after hypoxic injury. By electron microscopy, swollen neurons and dendrites, degenerating axons, disrupted myelin sheaths, and swollen astrocyte processes were observed in the nucleus tractus solitarius and ventrolateral medulla. Leakage of intravenously administered horseradish peroxidase was observed through vascular walls in hypoxic rats. These results suggest that increased VEGF and NO production in hypoxia resulted in increased vascular permeability, which, along with increased levels of glutamate, may have induced structural alterations of the neurons, dendrites, and axons. Administration of the antioxidant neurohormone melatonin (10mg/kg) before and after the hypoxia reduced VEGF, NO, and glutamate levels and improved ultrastructural abnormalities induced by hypoxia exposure, suggesting that it may have a therapeutic potential in reducing hypoxia-associated brainstem damage.

Neuronal connections of eye-dominance columns in the cat cerebral cortex after monocular deprivation

Plastic changes in intrahemisphere neuronal connections of the eye-dominance columns of cortical fields 17 and 18 were studied in monocularly deprived cats. The methodology consisted of microintophoretic administration of horseradish peroxidase into cortical columns and three-dimensional reconstruction of the areas of retrograde labeled cells. The eye dominance of columns was established, as were their coordinates in the projection of the visual field. In field 17, the horizontal connections of columns receiving inputs from the non-deprived eye via the crossed-over visual tracts were longer than the connections of the "non-crossed" columns of this eye and were longer than in normal conditions; the connections of the columns of the deprived eye were significantly reduced. Changes in the spatial organization of horizontal connections in field 17 were seen for the columns of the non-deprived eye (areas of labeled cells were rounder and the density of labeled cells in these areas were non-uniform). The longest horizontal connections in deprived cats were no longer than the lengths of these connections in cats with strabismus. It is suggested that the axon length of cells giving rise to the horizontal connections of cortical columns has a limit which is independent of visual stimulation during the critical period of development of the visual system.

Effect of Focused Ultrasound Applied With an Ultrasound Contrast Agent on the Tight Junctional Integrity of the Brain Microvascular Endothelium

Previous studies have investigated a potential method for targeted drug delivery in the central nervous system that uses focused ultrasound bursts combined with an ultrasound contrast agent to temporarily disrupt the blood-brain barrier (BBB). The purpose of this work was to investigate the integrity of the tight junctions (TJs) in rat brain microvessels after this BBB disruption. Ultrasound bursts (1.5-MHz) in combination with a gas contrast agent (Optison) was applied at two locations in the brain in 25 rats to induce BBB disruption. Using immunoelectron microscopy, the distributions of the TJ-specific transmembrane proteins occludin, claudin-1, claudin-5, and of submembranous ZO-1 were examined at 1, 2, 4, 6 and 24 h after sonication. A quantitative evaluation of the protein expression was made by counting the number of immunosignals per micrometer in the junctional clefts. BBB disruption at the sonicated locations was confirmed by the leakage of i.v. administered horseradish peroxidase (HRP, m.w. 40,000 Da) and lanthanum chloride (La 3+, m.w. ∼ 139 Da). Leakage of these agents was observed at 1 and 2 h and, in a few vessels, at 4 h after ultrasound application. These changes were paralleled by the apparent disintegration of the TJ complexes, as evidenced by the redistribution and loss of the immunosignals for occludin, claudin-5 and ZO-1. Claudin-1 seemed less involved. At 6 and 24 h after sonication, no HRP or lanthanum leakage was observed and the barrier function of the TJs, as indicated by the localization and density of immunosignals, appeared to be completely restored. This study provides the first direct evidence that ultrasound bursts combined with a gas contrast agent cause disassembling of the TJ molecular structure, leading to loss of the junctional barrier functions in brain microvessels. The BBB disruption appears to last up to 4 h after sonication and permits the paracellular passage of agents with molecular weights up to at least 40 kDa. These promising features can be exploited in the future development of this method that could enable the delivery of drugs, antibodies or genes to targeted locations in the brain. (E-mail: njm@bwh.harvard.edu)

Eye-rotation-induced spatial reorganization of horizontal connections in field 17 of the cat cortex

Six cats with rotation of one or both eyes (strabismus) produced surgically in the early postnatal period demonstrated torsional deviation of the eyes by 10-20 degrees in addition to the rotation. The spatial distribution of retrograde labeled neurons in field 17 was studied by microiontophoretic administration of horseradish peroxidase into individual cortical columns in fields 17 and 18. These studies showed that rotation of the eyes increased the extent of horizontal neuronal connections in field 17 along the projection of the vertical meridian of the field of vision. It is suggested that this reorganization of neuronal connections may support functional changes compensating for eye rotation, as described in the literature.

Limited uptake of foreign materials by resident macrophages in murine ovarian tissues

In the present study, the distributions of exogenously administered horseradish peroxidase (HRP) and colloidal carbon in gonadal tissues were observed in both male and female mice using light microscopy. HRP was injected intravenously and colloidal carbon was directly injected into the gonadal parenchyma. Thereafter, the gonads were obtained for histological examination. The results showed that staining of HRP and carbon was detected in the ovaries at a low level. In contrast, much staining was observed in the interstitial cells of the testes for a long period. This suggests that ovarian tissues are less active in the uptake of exogenous materials than testicular tissues in vivo. However, immunohistochemical examination using anti-macrophage antibodies revealed that the ovaries contained a large number of macrophages, as did the testes, under normal conditions. Therefore, the results indicate that resident macrophages in the ovaries exhibit weak endocytic activity of foreign materials in vivo.

Nonperfusion of retina and choroid in transgenic mouse models of sickle cell disease

Purpose. To determine if vascular occlusion and nonperfusion is associated with the outer retinal atrophy, retinopathy, and choroidopathy (chorioretinopathy) that occurs in the αHβS[βMDD] and αHβS[αMDβMDD] transgenic mouse models of sickle cell disease.Methods. Mice from the αHβS[βMDD] and αHβS[αMDβMDD] transgenic mouse lines that express high levels of human βS globin were anesthetized and administered horseradish peroxidase (HRP) intracardially. After 1 min, the animals were sacrificed, and the retina from one eye was excised, fixed, and developed in diaminobenzidine (DAB). The contralateral eye was fixed, embedded whole in glycol methacrylate, and HRP developed in 2.5 μm sections.Results. HRP reaction product (HRP-RP) and stained erythrocytes (RBCs) (due to endogenous peroxidase) were diffusely distributed within all vascular lumens in fiatmount retinas from control animals (littermates homozygous for the mouse βMajor deletion not expressing the βS transgene). In 42.5% of the transgenic mice expressing βS without any proliferative retinopathy, many blood vessels contained RBC plugs and lacked lumenal HRP-RP. In addition to packed RBCs, fibrin was sometimes present at sites of occlusion. In sections from whole eyes of the same animals, foci of photoreceptor degeneration were associated with areas of choriocapillaris nonperfusion (lumen that lacked HRP-PR). In areas with normal photoreceptors, the choriocapillaris appeared perfused (HRP-RP was present). In animals with proliferative chorioretinopathy, some neovascular formations lacked luminal HRP-RP, suggesting autoinfarction.Conclusions. Nonperfused retinal and choroidal vessels were observed in mice from the αHβS[βMDD] and αHβS[αMDβMDD] lines without retinal and choroidal neovascularization, whereas, all mice with neovascularization had nonperfused areas. Furthermore, small foci of PR loss were associated with areas of nonperfused choriocapillaris. These results suggest that sickle cell-mediated vaso-occlusions are an initial event in the chorioretinopathy and outer retinal atrophy that occurs in these models. Curr. Eye Res. 17: 438–444, 1998.

Evaluation of alternative proximal gastric vagotomy techniques after a 9-month interval in a rat model

Proximal gastric vagotomy (PGV) is an accepted operation for patients with ulcers that are refractory to medical management. Results comparable to those of standard, operative PGV have previously been demonstrated using endoscopic chemoneurolytic injection or laparoscopic laser seromyotomy in a porcine model. In this study, we evaluated several PGV techniques in regard to long-term effects on acid secretion, ulcer prophylaxis, and permanent vagal denervation in a rat model. Trans-mucosal injection of chemoneurolytic agents (cobaltous chloride, benzalkonium chloride, and phenol) and seromyotomy by CO 2 laser were performed. After 9 months, all rats received sub-serosal gastric injections of horseradish peroxidase (HRP) during laparotomy. Twenty-four hours later, an ulcerogenic dose of pentagastrin was administered sub-cutaneously. Three days after administration of HRP (to allow time for retrograde axonal transport and labeling of cells of the dorsal vagal nucleus with HRP), necropsy was performed. The pre-pyloric gastric mucosa was inspected for ulcerogenic changes, and a Congo red solution was applied to the gastric mucosa to map the acid-secreting areas. All PGV methods significantly diminished pentagastrin-induced ulceration when compared to sham controls. Benzalkonium chloride chemoneurolytic and laser methods were most effective for decreasing the size of acid-secreting areas. A reduced number of HRP-stained cells in the dorsal vagal nucleus indicated permanent denervation of vagal-gastric connections by operative and laser techniques. (Gastrointest Endosc 1994;40:316-20.)

Blue-light-induced Dysfunction of the Blood-Retinal Barrier at the Pigment Epithelium in Albino Versus Pigmented Rabbits

The purpose of this study was to determine the role of epithelial melanin in blue light phototoxicity of the retina. The first manifestation of the phototoxicity has been shown to be a breakdown of the blood-retinal barrier at the retinal pigment epithelium.The blood-retinal barrier function of six New Zealand albino rabbits was compared to that of four pigmented chinchilla rabbits after exposure to broad-band blue light (400-520 nm). Additionally, the spectral sensitivity of blood-retinal barrier dysfunction was determined by exposing 15 New Zealand albino rabbits to narrow-band blue light with peak intensity at λ = 408 nm, 418 nm, 439 nm, 455 nm and 485 nm (bandwidth: 11·7-13·5 nm). The blood-retinal barrier function was evaluated with vitreous fluorophotometry. Ultrastructural changes and permeability of the retinal pigment epithelium for horseradish peroxidase were evaluated in the albino rabbits with electron microscopy.Exposure to broad-band blue light up to 832 J cm-2 demonstrated the blood-retinal barrier of albino and pigmented rabbits to be equally sensitive.Electron microscopy of albino rabbits exposed to above-threshold energy demonstrated an increase of inclusion bodies in the retinal pigment epithelium and vacuolation of the cytoplasm. Transcellular passage of intra-arterially administered horseradish peroxidase through the pigment epithelium into the subretinal space was seen.The narrow-band exposures demonstrated that light of 439 nm was more effective than the light of other wavelengths in inducing barrier dysfunction in albino rabbits. This implies that chromophores absorbing at 439 ± 6 nm were responsible for the phototoxicity in albino rabbits.The results indicate that melanin does not have a damaging nor a protective role in phototoxicity since (1) the presence of melanin is not essential for blue-light-induced photochemical damage to the blood-retinal barrier at the retinal pigment epithelium, and (2) protection from this sort of damage is not greater in melanin containing epithelia than in non-melanin containing epithelia.

Fetal neocortical transplants in rats: blood-brain barrier development and partial permeability to IgG in long-surviving grafts

In previous work, allografts of fetal (E13-16) neocortical tissue transplanted into neocortical lesion cavities in newborn (PND 0-1) rats developed an impermeable blood-brain barrier (BBB) as shown by intravascular administration of horseradish peroxidase and by immunohistochemical staining for endogenous IgG. The present study examines the time course for the formation of the graft BBB by staining for endogenous IgG and also looks at transplants with extended survival times of 1-2 years. At two weeks post-transplantation, the grafts of all ten animals of this group showed evidence of IgG immunoreactivity within the graft parenchyma. This was greatest at the pial surface and adjacent to the ventricular surface of the transplant. By three weeks after transplantation, only four of nine grafts showed graft vessel permeability and this was confined to the area under the pia. At four weeks survival, one of nine grafts showed a small rostral patch of IgG reactivity, and a second animal showed very light, diffuse transplant labeling. The remaining seven animals were devoid of transplant IgG reactivity. At 11.5-28 months, three out of seven grafts had reaction product in the graft, indicating BBB permeability to IgG. In two of these older transplants, permeability was confined to the area around larger blood vessels, while one additional animal (28 months) showed dense labeling immediately below the pial surface. As in normal rats, host brain labeling was only found in circumventricular organs. These results show that circulating macromolecules are excluded from most CNS grafts within the first month of transplantation. This process progresses from the center of grafts and requires the longest time to complete in subpial regions possibly associated with healing of the pia. Some older grafts show leakage of protein which may relate to aging of the transplant or to a low-level graft rejection.

Vacuoles and vesicles in the rat junctional epithelium: A study with serial ultrathin sections

To investigate whether the electron-lucent structures resembling vesicles and vacuoles in the rat molar junctional epithelium (JE) are in fact intracellular or extracellular, a study using serial ultrathin sections was carried out. In one series of experiments, the animals were not treated before the tissues were conventionally fixed; in another, anesthetized animals were administered horseradish peroxidase 20 min before the tissues were fixed. A large number of electron-lucent structures resembling vesicles and vacuoles were detected in both the peripheral and central cytoplasm of the JE localized at enamel and connective tissue sites. These were 70 to 800 nm in diameter and had a lucency similar to that of the extracellular space in untreated specimens fixed with conventional fixative. Serial ultrathin sectioning revealed that the electron-lucent structures gradually became part of the extracellular space in the following sections. There were also found in the middle portion of the cytoplasm in specimens pretreated with horseradish peroxidase. Numerous vacuole-like structures containing peroxidase-positive materials were found to be contiguous with the extracellular space. A small number of vesicles, also containing peroxidase-positive materials, did not appear in the previous or following sections. These results indicate that almost all electron-lucent structures resembling vesicles and vacuoles in the JE are located at the end of a long infolding, and are still in contact with the extracellular space.

Explanation for the labeling of cervical motoneurons in young rats following the introduction of horseradish peroxidase into the calf

This study was carried out to determine whether cervical motoneurons, labeled following the introduction of horseradish peroxidase into the rat hind leg, belong to the cutaneous trunci motoneuron pool. The cutaneous trunci is a superficial muscle that extends from the axilla, over the flank, and into the thigh. Its nerve supply is derived from the brachial plexus. In experimental animals, horseradish peroxidase was either injected directly into the right gastrocnemius muscles, or applied to gelfoam and implanted over the calf muscles in the right leg of 5-, 10-, 15-day-old and adult rats. In control animals the cutaneous trunci was denervated prior to the administration of horseradish peroxidase. Labeled cervical motoneurons were present in the 5-, 10-, and 15-day-old but not the adult experimental groups and were located within the predetermined confines of the cutaneous trunci motoneuron pool. No labeling of cervical motoneurons was observed in any of the control groups in which the cutaneous trunci muscle was denervated. The most likely explanation for the labeling of cervical motoneurons in young rats was the local diffusion of horseradish peroxidase from the calf to the thigh, where it entered the cutaneous trunci muscle and was taken up by some of its motoneurons. The absence of such labeling in adult rats was probably due to the presence of connective tissue barriers to diffusion and to the greater distance between the site of horseradish peroxidase application and the cutaneous trunci muscle, which prevented the tracer from reaching the cutaneous trunci muscle and labeling its motoneurons.

Studies on the vasculogenesis in rat cerebral cortex.

The cerebral cortices are nourished by blood circulating through a capillary network branching from parent arteries. In this paper, vasculogenesis of the cerebral cortices in developing rats (11-18 days after birth) was studied by light and electron microscopy after intravenous administration of horseradish peroxidase (HRP). In the first phase of vasculogenesis, tentacles grew out from the distal end of the vascular cord (tip cells), and in the second phase, they extended to the afferent blood vessel. The reaction product of HRP was distributed not only in the primitive vascular lumen, but also in the peripheral extravascular space of the vascular cord. Most tentacles were free from reaction product, but, in some cases where reaction product was detected, the intercellular spaces had spread into the tip cells and reached the root of the tentacles. After the tentacles contacted the afferent vascular cord, the two vascular cords approached and fused with each other. Blood serum was able to circulate between the two vascular cords via the irregular interstices formed among the endothelial cells, cytoplasmic processes, and tentacles. Later, the primitive vascular lumen developed to a sufficient size to allow for the circulation of blood cells. The sequential events in the process of fusion between two vascular cords are illustrated. The following two points were also noticed in this study: 1) during cerebral vasculogenesis, degeneration and vacuolization in primitive endothelial cells occurred only in some of the serial sections and did not play a significant role in the formation of vascular lumen, and 2) cerebral pericytal macrophages (CPM) (classified as fluorescent granular perithelial cells by the authors) appeared close to the vascular cord, although the biological meaning of it remained unknown in the present investigation.

Neocortical transplants grafted into the newborn rat brain demonstrate a blood-brain barrier to macromolecules

Vascular permeability was examined in fetal neocortical transplants grafted into the cerebral cortex of newborn rats. Methods based on the histochemical labeling of intravenously administered horseradish peroxidase or on the immunocytochemical demonstration of endogenous immunoglobulin showed the presence of a blood barrier within the transplants.

The carotid sinus connections: a WGA-HRP study in the cat

The neural connections of the carotid sinus were studied by administration of horseradish peroxidase or a lectin conjugate to the adventitia of the carotid sinus of cats. The carotid sinus afferents project exclusively to the nucleus of the tractus solitarius (NTS). Rostral to the obex the projection is mainly ipsilateral with a strong contralateral component caudal to the obex. The carotid sinus projects to several NTS territories that do not receive afferents from the carotid body chemoreceptors; they are: the dorsolateral, the lateral extension of the commissural, the caudal intermediate, the ventrolateral and the gelatinosus subnuclei. In addition the carotid sinus central representation includes territories occupied also by carotid body terminals: dorsal, interstitial, rostral intermediate, medial and the medial part of commissural subnuclei (see previous paper). Labeled cell bodies were found in the petrosal (216 ± 37,mean±S.E.M.) nodose (825 ± 434) and superior cervical ganglia (3583 ± 1227) demonstrating the sympathetic efferent innervation of the carotid sinus and a dual sensory innervation via both the glossopharyngeal and vagus nerves.

Morphologic studies of acute rat cardiac allograft rejection across an isolated major histocompatibility complex class I (RT1A) disparity.

To define the morphologic correlates of acute rat cardiac allograft rejection across an isolated major histocompatibility complex (MHC) class I disparity, rejecting PVG.R1 cardiac allografts transplanted to (PVG x WF)F1 recipients were studied from days 4-8 posttransplantation. Documented ultrastructural tracer techniques as well as immunohistologic and immunoelectron microscopic methods were employed for morphologic analysis. Using intravenously administered horseradish peroxidase as a tracer probe for cell membrane permeability dysfunction, it was shown that severe diffuse loss of integrity of the microvascular endothelium preceded functional rejection, providing strong evidence that the allograft microcirculation is a central target of graft destruction. Also, rejection was associated with localized cardiac myofiber alterations prior to development of significant endothelial changes, indicating that cardiac muscle cells are additional cellular targets of immunologic injury. The ultrastructural features of progressive endothelial and myofiber injury, the predominance of MRC OX8+ lymphocytes and MRC OX6+ macrophages sequestered within the grafts, and the pattern of donor class I expression by allograft endothelium and cardiac myofibers were similar to those observed in rejecting allografts in full MHC-disparate combinations. Since it has been previously shown that MRC OX8+ class I-reactive T cells are absolutely required for rejection in this isolated class I-disparate model, the morphologic data raise the possibility that the OX8+ T lymphocyte subpopulation may also play a highly significant role in rat cardiac allograft rejection across a full MHC disparity.

Absorption of intact proteins by the intestinal epithelium of trout, Salmo gairdneri. A luminescence enzyme immunoassay and cytochemical study.

In the rainbow trout, a carnivorous species with a stomach, orally administered horseradish peroxidase (HRP) crossed the intestinal epithelium into the circulation. The protein first appeared in the blood 7 to 8 h after intubation, and could be assayed up to 24 h after administration. The method used, which combines ELISA (enzyme-linked immunoabsorbant assay) and chemiluminescence, enabled the transfer to be measured quantitatively. There was a direct correlation between the quantity ingested and the quantity transferred to the plasma within the experimental limits chosen. The clearance was monophasic and exponential (clearance rate: 3% per minute). Up to 6% of the ingested HRP was transferred to the blood. By cytochemistry it was possible to demonstrate that the protein crossed the intestinal cells at the level of the posterior segment, escaping the particularly intense intracellular lysosomal digestion. After entering the intercellular space, HRP was transferred to the interstitial space of the subepithelial lamina propria. During this transfer the HRP was in close contact with infiltrated macrophages and leukocytes resembling lymphoid cells. Thus, the passage of these protein particles could be the first indispensable step in the possible triggering of a local and/or a systemic immune response.

Peroxidase activity at nodes of ranvier in lumbosacral ventral spinal roots and in the PNS-CNS transitional region after intramuscular administration of horseradish peroxidase.

The axoplasm of nodes of Ranvier in feline lumbosacral ventral spinal roots was analysed by light and electron microscopy 18-168 h after the injection of horseradish peroxidase (HRP) into the medial gastrocnemius muscle. Three main HRP distribution patterns were distinguished at PNS nodes (bordered by Schwann cells only) of large fibres transporting HRP. The type A pattern, characterized by a distal accumulation of HRP-positive bodies and a proximal system of vesiculotubular membrane profiles. The incidence of this type of node was highest at relatively short survival times. The type B pattern, which appeared somewhat later, resembled the type A node with the addition of a disc-like, proximal accumulation of HRP activity. The type C pattern which contained scattered HRP-positive bodies and delicate strands of membraneous profiles, dominated 72 h after injection. The number of HRP-positive PNS-CNS borderline nodes (bordered by both Schwann cells and glial cells) was less than 5% of the corresponding value in the same fibres of the ventral root proper. A highly segregated state of the axoplasm of PNS-CNS borderline nodes was noted only in two cases. The observations indicate a functional difference between nodal axoplasm at the PNS-CNS borderline and nodal axoplasm in the PNS part of the alpha motor neuron.

Mechanisms of blood-brain barrier breakdown after microembolization of the cat's brain.

Unilateral microembolization of the cat brain with carbonized microspheres 15 microns in diameter ten minutes (min) before death induced multifocal disruption of the blood-brain barrier (BBB) to horseradish peroxidase (HRP) in nine adult cats. The gross pattern of HRP extravasates was studied: (a) after five min of in vivo circulation, (b) following infusion of HRP immediately before chemical fixation, and (c) following infusion of HRP after 60 min of aldehyde fixation. Examination of the material from the three different experimental groups revealed no qualitative differences at the light microscopic level; specific features such as ring-shaped extravasations of HRP occurred irrespective of the mode of tracer injection. Tracer-filled pinocytotic vesicles and tubular profiles were abundant in the vascular endothelium after in vivo circulation of HRP, but were virtually absent after supravital and postmortem HRP administration. The results suggest that BBB breakdown for proteins after microembolization is not an energy-dependent process mediated by either pinocytosis or tubular-endothelial channel formation.