Horseradish Peroxidase Permeability Research Articles

Topographical mapping of sites of enhanced HRP permeability in the normal rabbit aorta.

The spatial distribution of sites of enhanced permeability to the macromolecule horseradish peroxidase (HRP) in the normal rabbit aorta after one min circulation was studied using image analysis. These sites, referred to as "HRP spots," exhibit a nonuniform distribution that is qualitatively similar in all rabbits studied. The density of HRP spots is highest in the aortic arch, decreases distally, reaches a minimum in the lower descending thoracic aorta, and then increases again in the abdominal aorta. The region of highest spot density follows a clockwise helical pattern in the aortic arch and outside the arch occurs in streaks largely oriented in the bulk flow direction. The streaks in the abdominal aorta localize along the anatomical right lateral wall and occasionally along the left lateral wall proximal to the celiac artery and along the ventral wall between the celiac and superior mesenteric arteries. The density of spots is high in the immediate vicinity of aortic ostia with the most elevated density being distal to ostia in most cases. At a short distance from the ostium edge of the celiac and superior mesenteric branches the proximal density is comparably high, and no preferred spot orientation is observed around the brachiocephalic vessel. These results are consistent with an influence of localizing factors such as detailed hemodynamic phenomena and/or arterial wall structural and/or functional variations.

Lower limb ischaemia and reperfusion alters gut permeability

The gut may be important in the aetiology of multiple organ failure (MOF) by amplifying of the inflammatory response to trauma. We investigated the effects of the reperfusion of ischaemic lower limbs on gut permeability. Male Wistar rats (n = 30) were randomised to (group 1) controls; (group 2) 3 h bilateral hind-limb ischaemia alone; or 3 h ischaemia followed by (group 3) 15 min reperfusion or (group 4) 2 h reperfusion. Gut permeability and plasma endotoxin were measured prior to tourniquet application, immediately before tourniquet release, and following reperfusion. To evaluate the effect of the hypotension that follows tourniquet release, (group 5) sodium nitroprusside was infused in further controls to maintain mean arterial pressure (MAP) at 75 mmHg for 2 h. Horseradish peroxidase was instilled into the isolated ileo-caecal loop 15 min before the animals were killed to measure permeability of horseradish peroxidase through mucosal intercellular tight junctions by electron microscopy. Mean arterial pressure increased from 105 +/- 5 mmHg to 136 +/- 4 mmHg on tourniquet application and fell to 79 +/- 7 mmHg following reperfusion (p less than 0.05). In group 1 (controls), group 2 (ischaemia alone animals) and group 5 (ischaemia and nitroprusside) one animal out of six demonstrated permeability to horseradish peroxidase. Following reperfusion, horseradish peroxidase permeability had not developed by 15 min (group 3) but was present in all animals by 2h (group 4) (p = 0.015 Fisher's exact test). Plasma endotoxin increased from 21.8 +/- 2.0 pg ml-1 to 30.7 +/- 2.6 pg ml-1 following 2 h reperfusion (p less than 0.05 Scheffe F-test).(ABSTRACT TRUNCATED AT 250 WORDS)

Dysfunction and repair of the blood-retina barrier following white light exposure: A fluorophotometric and histologic study

The purpose of this study was to pinpoint the site of blood-retina barrier disruption after white light exposure and determine the course of barrier repair. The retinas of 25 anaesthetized pigmented rabbits were exposed for 1 hr to the light of a xenon arc lamp filtered to eliminate ultraviolet and infrared light. The light intensities selected were near the threshold intensity causing visible retinal lesions in order to evaluate the function of the blood-retina barrier (BRB) in this range. Functional assessment of the BRB was made with vitreous fluorophotometry (VF), and electron microscopy (EM) after intra-arterial administration of horseradish peroxidase (HRP) as tracer. In 11 of the 14 rabbits exposed to threshold intensity (90–110 mW cm −2; retinal field of illumination, 0·64 cm 2), a breakdown of the BRB was demonstrated by a 2–40-fold increase in the permeability of the BRB for fluorescein and by transcellular passage of HRP through the retina pigment epithelium (RPE). All 11 rabbits developed oedematous fundus lesions. Within a week, pigmentary alterations of the fundus were seen on ophthalmoscopy, while the BRB permeability for fluorescein and HRP had returned to normal. EM of the retina showed slight swelling of RPE during the period of increased permeability but no alterations of the neuroretina. After functional barrier repair, the RPE cells demonstrated irregularity of the melanin pigment alignment and some loss of the monocellular arrangement. In six rabbits exposed to subthreshold light intensity (65–89 mW cm −2) no fundus lesion developed and EM evaluation of the BRB was normal. All five superthreshold exposed rabbits (111–140 mW cm −2) developed ophthalmoscopically visible lesions, and VF and EM evidence of BRB permeability increase. In the range 65–140 mW cm −2, fluorescein leakage into the vitreous after a 1-hr exposure was found to increase exponentially with the light intensity starting at a value of 69 mW cm −2 (corresponding with 250 J cm −2; 95% confidence intervals, 200 and 280 J cm −2). The results show that low-intensity white light primarily affects the RPE cells in rabbit eyes inducing a temporary breakdown of the BRB. This functional disturbance can be quantified with fluorophotometry prior to the development of morphological or ophthalmoscopic changes. Functionally, the barrier recovers although morphologically, on ophthalmoscopy and EM, the retina remains altered.

Augmented polyol pathway activity and retinal pigment epithelial permeability in the diabetic BB rat

In the present study we investigated the relationship between an augmented polyol pathway and the breakdown of the blood-retinal barrier in the spontaneously diabetic BB rat. Permeability experiments were performed in diabetic and age-matched non-diabetic BB rats in a longitudinal fashion using horseradish peroxidase. Increased permeability of horseradish peroxidase across the retinal pigment epithelium was noted after 6 months of diabetes. Abnormalities of the basal plasmalemmal infoldings of the retinal pigment epithelium were noted in the control animals and appeared to be exaggerated in diabetic rats. Simultaneous quantitative ultrastructural immunohistochemistry, using an affinity purified anti-BB rat aldose reductase antibody and protein-A gold, revealed a significant increase in the aldose reductase immunoreactivity of the retinal pigment epithelium in diabetic animals. These findings suggest that an augmented polyol pathway activity may play a role in the pathogenesis of the blood-retinal barrier breakdown at the level of the retinal pigment epithelium in the diabetic BB rat.

Time course of the blood-arterial wall barrier disruption following experimental subarachnoid haemorrhage.

The time course of the blood-arterial wall barrier disruption following experimental subarachnoid haemorrhage (SAH) was studied in 24 rabbits. Animals with SAH received two successive blood injections through the cisterna magna. Horseradish peroxidase (HRP) was given intravenously 30 minutes before sacrifice to assess the integrity of the barrier. In the basilar arteries taken from animals that were sacrificed 4 days after the first SAH, HRP-reaction products were diffusely observed in the subendothelial space. Three weeks following the first SAH, permeation of HRP was still observed in half of the animals. However, in animals sacrificed 7 weeks after the first SAH, no permeation of HRP into the subendothelial space was noted. Opening of the interendothelial space seemed to be the major mechanism for HRP permeation into the subendothelial space rather than transendothelial vesicular transport. Disruption of the blood-arterial wall barrier in the major cerebral arteries following SAH may play a role in the pathogenesis of vasospasm.

Histopathological and blood-brain barrier changes in rats induced by an intracerebral injection of human recombinant interleukin 2.

Adoptive immunotherapy utilizing human recombinant interleukin 2 (rIL-2) in conjunction with lymphokine-activated killer cells has shown some efficacy in the treatment of various types of cancers, particularly renal carcinoma and melanoma. Intravenous administration of rIL-2, with or without lymphokine-activated killer cells, produces a variety of serious side effects and approximately one-third of the patients experience a decrease in neurological status. Our previous investigations in animals have indicated that a single intravenous injection of rIL-2 can compromise the integrity of the blood-brain barrier (BBB). The present study examined the histopathological effect and BBB changes in rats which occur after a single intracerebral injection of rIL-2, its excipient, or saline into the parietal lobe. Animals were killed at various intervals up to 8 days (1 hour after intravenous injection of horseradish peroxidase), and the brain tissue was sectioned and processed for light microscopy. All animals showed increased cerebrovascular permeability for horseradish peroxidase due to traumatic BBB disruption at 4, 12, and 24 hours after injection. Extravasation of horseradish peroxidase persisted at 3 and 8 days only in animals injected with rIL-2. Injections of rIL-2 led to an increased leukocytic infiltration into the injection site, perivascular cuffing, and localized edema by 24 hours, which continued to increase over the 8-day study period. These results suggest that a single injection of human rIL-2 into the brain of rats induces an influx of leukocytes into the brain and may contribute to the cellular events that perpetuate a trauma-induced compromise in the integrity of the BBB.

Densitometric measurement of increased endothelial permeability in arteriosclerotic plaques and inhibition of permeability under the influence of two calcium antagonists

A densitometric technique was established to investigate quantitative changes in endothelial permeability for horseradish peroxidase (HRP), mol. wt. 40000 daltons, in rabbit carotid artery. Repeated weak electrical stimulations of rabbit carotid arterial walls with implanted electrodes lead to fibromuscular plaques mainly beneath the anode. It could be demonstrated that there exists a typical growth curve of the plaques dependent on the number of days of electrostimulation, with a fast proliferation rate of smooth muscle cells in the first 2 weeks of electrostimulation, and an increasing retardation of proliferation during the next 4 weeks. Endothelial permeability for HRP increases in close relation to the plaque development. Intravenous applications of single doses of the calcium entry blockers flunarizine or nimodipine are able to inhibit the increased permeability of the endothelial lining covering arteriosclerotic plaques. The intensity of the inhibitory action of these calcium antagonists correlates with the size of the arteriosclerotic plaques in inverse proportion, but nevertheless in large plaques an inhibitory effect is seen.

ラット胎盤における栄養膜のHorseradish peroxidaseの透過性

ラット胎盤における栄養膜のHorseradish peroxidaseの透過性

Antigen Absorption by the Jejunal Epithelium of Children with Cow's Milk Allergy

To establish if intestinal permeability to exogenous antigens is involved in cow's milk allergy (CMA) in infants, 33 children 1 to 24 months old (18 controls and 15 with CMA) were tested for intestinal permeability to the protein marker horseradish peroxidase (HRP). Jejunal biopsies were performed either during the initial period of diagnosis, at the mean (and SE) age of 3 +/- 1 months, and/or 1 yr later, at the age of 13 +/- 2 months, just before and after a milk challenge. A small fragment of the biopsy was studied for histology and the remainder was mounted in an Ussing chamber for simultaneous measurement of mucosal to serosal transport of HRP in its intact and degraded forms and electrical parameters including short-circuit current and conductance. No modification in HRP absorption was noted in control children aged from 2 months to 11 yr, indicating that gut closure probably occurred earlier in life. During the initial period of CMA, transepithelial HRP fluxes were significantly higher, about 8-fold, in children with CMA (intact HRP flux = 48.5 +/- 15.2, 95% confidence interval, 11.2 to 85.7 versus 5.9 +/- 1.2, 95% confidence interval 2.9 to 8.3, in control children). In addition, short-circuit current was increased but conductance was unchanged. After several months on a milk-free diet, HRP flux and short-circuit current returned to control values. Just after the milk challenge and independently of the clinical issue, a slight rise in HRP permeability was observed but it was not significant and remained within control values.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of pentoxifylline on endothelial permeability of rabbit carotid artery wall

Weak electrical stimulation of the rabbit carotid artery wall with direct current impulses over a period of 45 min increased the endothelial permeability for horseradish peroxidase (HRP), molecular weight 40 000 Da, beneath the anode region of the electrodes. The stimuli caused a massive accumulation of the reaction products of peroxidase in the subendothelial space. With a microdensitometric technique, it was possible to quantify the amount of the peroxidase reaction products in the subendothelium, and to compare the amounts in the non-stimulated regions with those in the stimulated regions. Intravenous administration of pentoxifylline before stimulation inhibited the transendothelial transport of peroxidase. Inhibition was pronounced in the stimulated regions, whereas the uptake of HRP in the non-stimulated regions was only slightly lower than that in non-treated animals. It was demonstrated that the extent of inhibition was dose-dependent (3, 10 and 30 mg pentoxifylline/kg). Electron microscopic examination showed that the main route of permeation of HRP was through the interendothelial clefts. However, electrical stimulation also caused an increase in the vesicular uptake of HRP into endothelial cells. Both pathways seem to be influenced by pentoxifylline.

Compartments in the organum vasculosum laminae terminalis of the rat and their delineation against the outer cerebrospinal fluid-containing space

Using intravenously injected horseradish peroxidase (HRP) as tracer, we demonstrate, that--in contrast to other neurohemal regions--the organum vasculosum laminae terminalis (OVLT) is composed of two functionally different divisions. Both parts of the OVLT are endowed with fenestrated capillaries which, however, obviously differ in their permeability for HRP. In one of these portions the neurohemal region remains unlabeled under the experimental conditions used, while the other portion, in analogy to the majority of neurohemal regions, is labeled by the tracer. The functionally different divisions of the OVLT are separated from one another by tanycytic processes and meningeal cells establishing a barrier between the two hemal compartments. The meningeal elements penetrate the organ in the form of an uninterrupted layer; they are continuous with the pia mater and produce large amounts of basal lamina-like material. Furthermore, they provide the delineation of the OVLT against the outer cerebrospinal fluid-containing compartment, a structural feature that is characteristic of both divisions of the OVLT and corresponds to the arrangement of meninges in all other portions of the brain where a blood vessel penetrates its surface.

Intestinal absorption of macromolecules during viral enteritis: an experimental study on rotavirus-infected conventional and germ-free mice.

ABSTRACT: Epithelial transport and degradation of horseradish peroxidase (HRP), a macromolecular tracer, was studied in conventional and germ-free suckling mice following an experimental infection with rotavirus. Conventional and germ-free mice developed diarrhea from days 2 to 8 postinfection (pi), with growth failure. In mucosal homogenates, infectious virus detected by immunofluorescence on MA 104 cells was present from day 2 through day 8 pi in germ-free mice, but persisted longer (day 13 pi) in conventional mice. Only mild histological lesions were observed during diarrhea, but obvious macrovacuolation of epithelial cells and increased cellular density occurred during the convalescence period (days 9 to 13 pi). Intact and degraded HRP fluxes from mucosa to serosa were measured in vitro on segments of jejunum mounted in Ussing chambers. Both groups of mice developed increased HRP permeability during the experimental period, but at different times after inoculation: during the diarrheal period (days 2 and 3 pi) conventional mouse epithelium absorbed five times more HRP than noninfected controls and during the convalescence period (days 9 to 13 pi) HRP absorption in germ-free mice rose 10-fold as compared to its level before infection. In both cases, this increase in HRP permeability was entirely due to an increase in intact HRP absorption, probably via a transcellular route, and occurred without any alteration in degraded HRP transport. These results indicate that in mice, rotavirus infection causes a transient rise in gut permeability to undegraded proteins. The intestinal microflora seems to affect the timing, magnitude, and duration of this increased permeability.

Formation and Regression of Capillary Sprouts in Corpora lutea of Immature Superstimulated Golden Hamsters

The ultrastructure of developing and regressing capillary sprouts was studied in corpora lutea of immature golden hamsters between days 4 and 7 after the application of serum gonadotrophin of pregnant mares (PMSG). Horseradish peroxidase (HRP), an endothelial tracer, was localized by ultrahistochemistry. The vascular permeability of HRP was quantified by an enzyme assay in ovarian homogenates. Sprouting endothelial cells looked activated. They showed micropinocytotic vesicles in a high endothelium surrounded by basal laminae. Early capillary growth was at its maximum on day 4 after PMSG. Advanced capillary growth was seen on days 4 and 5 after PMSG. The vascular lumina were formed by dilatation of the interendothelial space. Regression of capillary sprouts started on day 5, was most intense on day 6 and negligible on day 7. Two processes of regression were observed. One led to a complete destruction, the other to an incomplete one. Vascular permeability decreased between days 5 and 6 after PMSG. It is concluded that the corpus luteum can be viewed as a physiological model of angiogenesis.

Barrier disruption in the major cerebral arteries during the acute stage after experimental subarachnoid hemorrhage.

The effects of experimental subarachnoid hemorrhage (SAH) on the blood-arterial wall barrier in the basilar arteries were studied during the acute stage after SAH. SAH was induced in rats by injecting fresh autologous blood into the cisterna magna. Horseradish peroxidase (HRP) was given intravenously before killing the animals to assess the integrity of the barrier. In the basilar arteries taken from the animals that were killed 30 minutes after the cisternal injection of either mock cerebrospinal fluid or arterial blood, HRP reaction products were diffusely observed in the subendothelial spaces and smooth muscle layers. At 5 hours after the blood injection, no permeation of HRP into the subendothelial space was observed. Endothelial cell transcytosis seemed to be the important mechanism for HRP permeation into the subendothelial space rather than the opening of interendothelial junctions. The disruption of the blood-arterial wall barrier in the major cerebral arteries after SAH may be involved in the pathogenesis of vasospasm.

Altered Jejunal Permeability to Macromolecules During Viral Enteritis in the Piglet

We studied the macromolecular permeability of segments of jejunum from 2-wk-old piglets after the animals had been experimentally infected with an invasive enteric virus, transmissible gastroenteritis virus. Jejunal segments were mounted in Ussing chambers at stages of the infection, and permeability was measured using three probe molecules of differing molecular weights. In control tissue, permeability to horseradish peroxidase was 2.6 times higher across segments with Peyer's patches than across segments without Peyer's patches, whereas polyethylene glycol 4000 and mannitol permeabilities were the same in patch and nonpatch segments. Twelve hours after infection, when virus had invaded the mucosa causing a structural lesion, and before diarrhea had begun, horseradish peroxidase permeability increased in non-patch-containing segments to equal that across patch-containing tissue. At this early 12-h stage, polyethylene glycol 4000 and mannitol permeation were unchanged in patchcontaining segments compared with controls. Ninety-six hours after transmissible gastroenteritis infection, when diarrhea was severe, horseradish peroxidase permeability in patch-free segments had returned to normal and patch-containing tissue permeability was diminished below control levels. Increased macromolecular permeability appears to occur only in the very early invasive stage of this viral enteritis and only in patch-free segments. Any consideration of the immunologic relevance of these complex phenomena must take into account the specialized function of the Peyer's patch regions of the small intestine.

Permeability of different experimental brain tumor models to horseradish peroxidase.

The permeability of different brain tumor models to horseradish peroxidase (HRP) was examined by determining the fraction of tumor that contained HRP after intravenous administration. The intracerebral tumor models studied were Avian Sarcoma Virus (ASV)-induced tumors and tumors from transplanted RG-2, S69-C1-5, and 9L cell lines. The average fraction of RG-2 tumors permeable to HRP was .95; of S69-C1-5 tumors, .699; of ASV-induced tumors. .63; and of 9L tumors, .52. Except for the RG-2 tumors, there was considerable regional variation in HRP permeability, which was most marked in the ASV-induced tumors. In ASV-induced tumors, HRP permeability did not correlate with tumor histological classification, size, or anatomic location within the brain. The subcutaneous tumor models studied were RG-2-, S69-C1-5, and 9L-transplanted tumors in rats, and human glioblastoma cell lines transplanted into nude mice. All were completely permeable to HRP. These results indicate that significant differences in permeability to HRP exist among brain tumor models when the tumors are intracerebral, and that all subcutaneous tumors from transplanted glial cell lines are completely permeable to HRP. These variables must be considered in future studies of permeability in experimental brain tumors. Care must be exercised in extrapolating results about permeability from one brain tumor model to another.

Protein transport across cerebral vessels during metrazole-induced convulsions.

The effects of pentylenetetrazole-induced convulsions on cerebrovascular permeability for horseradish peroxidase were studied in locally anesthetized, artificially respired, paralyzed cats. Horseradish peroxidase circulated for 1, 2.5, 5, 10, 30, and 60 min and accumulated in medium-size, i.e., arterial or venous, blood vessel walls. Cerebral neuropil was permeated earliest in thalamic nuclei including medial and lateral geniculate nuclei. No tight junctions containing horseradish peroxidase were found. With 1.0 and 2.5 min circulation of horseradish peroxidase, arterial adventitia and basement membranes contained more peroxidase than surrounding interstitial space in contrast to veins and capillaries. Convulsions increased the number of endothelial vesicles containing horseradish peroxidase in arteries, veins, and capillaries but total number (stained plus unstained) of endothelial vesicles was increased slightly only in capillaries. We conclude that increased permeability is initially at the arterial level, although veins may also become permeable. The role of the endothelial vesicles in transit or protein in and out of the nervous system is not clear.

Transendothelial vesicular transport of protein in brain edema induced by ultraviolet irradiation.

Focal vasogenic brain edema was induced by ultraviolet irradiation of the exposed cerebral cortex of 16 cats. In 5 animals horseradish peroxidase was intravenously injected at times varying from 30 min to 24 h following irradiation and allowed to circulate for 45 min. Fixation was carried out by perfusion and immersion with glutaraldehyde. The tissue, part of it incubated for peroxidatic activity, was treated for electron microscopy. The UV-irradiation leads to a shallow coagulation of the superficial cortex from which a wide zone of edematous tissue spreads to the deep white matter within 24 h. Arterioles, capillaries and venules of this zone show enhanced pinocytotic activity and a concurrent rise in permeability for horseradish peroxidase which is found in micro- and macropinocytotic vesicles, in tubular vesicle-like structures, in endothelial wall invaginations and apparently not membrane-bound. These changes are most pronounced in venules which after 48 h allow penetration of reaction product though the base membrane into the surrounding neuropil. There is no evidence for the penetration of tight junctions which appear intact.

Permeability of rabbit corneal epithelium to horseradish peroxidase after the influence of benzalkonium chloride.

A tight barrier against permeation of horseradish peroxidase into the corneal epithelium exists at the corneal surface adjacent to the tear film. The present light and transmission electron microscopic study reveals that the cationic surfactant, benzalkonium chloride, which is commonly added to eye drops as a preservative, breaks down this barrier. Lysis of the cell membranes was demonstrated, resulting in a leakage of the tracer into and underneath the superficial cells. The grade of cellular destruction caused by benzalkonium chloride was dependent upon the concentration and exposure time of the drug.