Diffusion Of Horseradish Peroxidase Research Articles

Perivascular CSF flow in the rat cerebellum.

Cerebrospinal fluid (CSF) flow from the subarachnoid space into the cerebellum was studied in rats using Horseradish peroxidase (HRP) injected into the cisterna magna. Animals were sacrificed after 0, 10 or 30 min Transpial diffusion of HRP did not extend beyond the Purkinje cell layer. At all time intervals, perivascular spaces labelled with HRP were observed in cerebellar grey and white matter. In the cerebellar granular layer and white matter, HRP was usually present only in perivascular spaces and the surrounding extracellular space. There was no evidence of a preferential flow towards the fourth ventricle. These results suggest that there is a perivascular pathway for rapid flow of CSF from the subarachnoid space into the perivascular spaces and then into the cerebellar extracellular space. The Purkinje cell layer appears to act as a barrier to diffusion from the pial surface. Copyright 1999 Harcourt Publishers Ltd.

Disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators: a role for Rho/Rho kinase.

Disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators: a role for Rho/Rho kinase.

Perivascular CSF flow in the rat cerebellum

Cerebrospinal fluid (CSF) flow from the subarachnoid space into the cerebellum was studied in rats using Horseradish peroxidase (HRP) injected into the cisterna magna. Animals were sacrificed after 0, 10 or 30 min Transpial diffusion of HRP did not extend beyond the Purkinje cell layer. At all time intervals, perivascular spaces labelled with HRP were observed in cerebellar grey and white matter. In the cerebellar granular layer and white matter, HRP was usually present only in perivascular spaces and the surrounding extracellular space. There was no evidence of a preferential flow towards the fourth ventricle. These results suggest that there is a perivascular pathway for rapid flow of CSF from the subarachnoid space into the perivascular spaces and then into the cerebellar extracellular space. The Purkinje cell layer appears to act as a barrier to diffusion from the pial surface.

Tract-tracing in the nervous system of vertebrates using horseradish peroxidase and its conjugates: tracers, chromogens and stabilization for light and electron microscopy

Surprisingly the first generation of tract-tracing techniques based on intra-axonal transport, that is the methods utilizing the uptake and transport of horseradish peroxidase (HRP), still rank among the most widely used neuroanatomical tracing techniques. The success of these methods can be ascribed to several characteristics. They are fast and easy to implement. Complicated injection apparatus is unnecessary. The reaction products are visualized through simple histochemical reactions, and they are permanent or can be stabilized into permanency. Most usefully, the reaction products are visible in the light and electron microscopes. HRP (mol. wt. 44 kDa) is extracted from the roots of the horseradish plant ( Cochlearia armoracia L.). Uptake of HRP into cells occurs via a passive process of endocytosis [3]. Since lectins like wheat germ agglutinin (WGA) and bacterial toxin fragments (subunit B of cholera toxin (CTB)) [17, 32, 34]strongly induce active, receptor-mediated uptake mechanisms, conjugates of these substances with HRP have been successfully applied in sensitive tract-tracing [4, 8, 34, 35] HRP and its conjugates are transported both in anterograde and retrograde direction [19, 20]. Retrograde transport occurs in small vesicles that are incorporated in lysosome-like vacuoles and in the Golgi apparatus. These vesicles differ in membrane properties from the anterogradely transported HRP vesicles [3, 4, 17]. The retrogradely transported vesicles tend to fuse and thus accumulate HRP at high densities, facilitating the visibility of the final reaction product. The anterogradely transported HRP does not accumulate directly in lysosome-like bodies and is distributed diffusely and therefore often requires specific visualization methods [5, 8, 10, 12, 31]. HRP and WGA-HRP may therefore be used in anterograde and retrograde transneuronal (multineuron) transport studies [15, 19]. Even in fixed material, labeling through diffusion of HRP can provide details on neural connections [18]. Visualization of transported HRP is achieved by means of using the oxidative enzymatic activity of HRP to precipitate a chromogen according to the following reaction: HRP+H 2O 2⇄[HRP.H 2O 2] [HRP.H 2O 2]+(chromogen)H 2⇄HRP+2H 2O 2+chromogen precipitate The final reaction product may be soluble in buffer or ethanol and may require stabilization to prevent fading. In this protocol we discuss the widely used chromogens 3,3′-diaminobenzidine tetrahydrochloride (DAB), 3,3′,5,5′-tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC) and p-phenylenediamine dihydrochloride with pyrocatechol (PPD-PC). Other possible chromogens, not discussed here, are 4-chloro-1-naphthol (4C1N), 3-amino-9-ethylcarbazole (AEC) and o-phenylenediamine (OPD). The visualization of the reaction product can be further improved by intensification with metal salts. At the light microscopic level (LM) this intensification enables color differentiation between distinct markers [1, 14, 23]. In the present protocol we provide an up-to-date guideline for the application of HRP and its conjugates in tracing with special emphasis on electron microscopic (EM) visualization. Some modifications for stabilization and of metal intensification to enhance visibility are incorporated in conjunction with specific methods for multiple labeling in combined tract-tracing experiments.

Acute effects of human recombinant tumor necrosis factor-? on the cerebral vasculature of the rat in both normal brain and in an experimental glioma model

The effects of intravenous (IV) infusion of human recombinant tumor necrosis factor-alpha (rTNF-alpha, Cetus) on normal brain and malignant glioma in rats were examined. Twelve Fischer 344 rats were given either a single injection of 10(6) U rTNF-alpha or injections of 5 x 10(5) U rTNF-alpha for three days. One day post-rTNF-alpha injection(s), rats were injected IV with horseradish peroxidase (HRP) to determine blood-brain barrier (BBB) breakdown and, one hour later, were perfused with an aldehyde fixative and processed for histologic examination. Treatment of normal rats with rTNF-alpha by either dosage or schedule caused no remarkable histopathologic changes in the brain and no alteration in BBB integrity. Human glioma models were produced by intracerebal inoculation of 10(4) syngeneic RT-2 glioma cells into the right parietal lobe of 30 rats. Animals received single IV injections of 10(6) U human rTNF-alpha or its excipient (TNF-E) as above on day 3, 7, or 10 post-tumor inoculation or multiple injections of 5 x 10(5) U rTNF-alpha beginning on day 7, 10, or 12 post-tumor inoculation. With a single IV injection of either rTNF-alpha or its excipient, 3-day models showed a similar pattern of HRP extravasation limited to the extracellular space of the tumor inoculation site. In 7-day models treated with a single IV injection of rTNF-alpha or TNF-E, HRP extravasated throughout the tumor, but did not exceed peritumoral margins. In 10-day models treated with a single injection of TNF-E, HRP was found only in the tumor and immediate peritumoral regions while rTNF-alpha-treated rats showed more extensive areas of BBB breakdown with HRP evident throughout the entire right hemisphere and extending via the corpus callosum into the contralateral hemisphere. Pericapillary halos were also evident around the small blood vessels within the edematous areas of the corpus callosum. Within tumors, hemorrhagic necrosis and adherence of neutrophils to vessels was observed only in animals treated with rTNF-alpha at 10 days post-tumor inoculation. Multiple IV injections of rTNF-alpha in 7 and 10-day models triggered widespread hemorrhagic necrosis, neutrophil adherence and infiltration in the tumor. There was also extravasation and diffusion of HRP from the site of glioma into the contralateral hemisphere. Twelve-day models treated with multiple rTNF-alpha injections, in addition, showed irregular luminal surfaces and gaps between adjacent endothelial cells of tumor vasculature.(ABSTRACT TRUNCATED AT 400 WORDS)

The Permeability of Capillaries in the Eustachian Tube Mucosa

The transendothelial passage of horseradish peroxidase (HRP) and diffusion of HRP into the pericapillary space, injected intravenously into cats, was studied at the light and electron microscopic level in the mucosa of the eustachian tube. The permeability of capillaries in the pharyngeal two-thirds of the tube was higher than in the tympanic one-third though all the subepithelial capillaries were of the continuous type. In the lamina propria of the subepithelium in the pharyngeal two-thirds much HRP was found 10 min after its intravenous injection, but in the tympanic one-third, it was absent in the perivascular spaces. The capillaries of the pharyngeal two-thirds had many projections and marginal folds inside the capillary lumen and large pinocytotic vacuoles containing HRP as well as many micropinocytotic vesicles. On the other hand, the capillaries in the tympanic one-third had many micropinocytotic vesicles too, but only a few large pinocytotic vacuoles.

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.

Collateral branching in axonal projections to spinal cord from paramedian reticular nucleus neurons

Experiments were done in cats to identify neurons in the paramedian reticular nucleus (PRN) sending collateral axons to the region of the intermediolateral nucleus (IML) at different levels of the thoracic cord by using lectin-conjugated horseradish peroxidase (HRP) and double-labeling fluorochrome histochemistry to retrogradely label PRN neurons. Injections of Fast blue (FB) into the spinal cord at the T 2 level centered in the region of the IML were coupled with injections of Nuclear yellow (NY) into the ipsilateral cord at either the T 4 or T 7 level centered in the region of the IML. Neurons in the PRN retrogradely labeled after diffusion of HRP into the region of the IML at the T 2 level were observed througout the rostrocaudal extent of the ventral PRN. In addition, a few labeled neurons were noted in the ventral portion of the dorsal PRN. About 40% of the neurons in the PRN which were labeled with FB after an injection at the T 2 level were also labeled with NY injected into the cord in further caudal segments. These data suggest that the PRN may exert its influence on the cardiovascular system partly through collateral axonal branches to widely separated populations of sympathetic preganglionic neurons in different spinal segmental levels.

Labeling of motoneurons supplying the cutaneous maximus muscle in the rat, following injection of the triceps brachii muscle with horseradish peroxidase

Injection of the triceps brachii muscle of the rat with horseradish peroxidase (HRP) led to labeling of motoneurons in the dorsolateral and ventral aspects of the ventral grey horn of the brachial spinal cord. Ventral, labeled neurons were absent, however, in rats in which the motor branches of the radial nerve to triceps were exposed to HRP. Cutting the radial nerve, prior to the injection of the triceps, did not diminish this ventral labeling, whereas cutting both the radial nerve and the nerves to the cutaneous maximus muscle before injection completely eliminated it. Leakage or diffusion of HRP from the triceps and its subsequent uptake by damaged fibers of the cutaneous maximus muscle was the most likely cause of the labeling of the ventrally located motoneurons.

Anterograde and retrograde filling of central neuronal systems with horseradish peroxidase under in vitro conditions

In this study, a simple technique for neuronal tracing with horseradish peroxidase (HRP) in vitro is described. It can be applied to excised small brains of poikilothermic vertebrates, or to trimmed blocks or slices of brain tissue from homoiothermic vertebrates. The filling technique relies on the diffusion of HRP into transected axons of supravital neurons, and gives a Golgi-like staining of the filled neuronal profiles. The incubation is carried out at 4°C, which minimizes unspecific background staining caused by pinocytotic uptake of HRP. The technique was tested for visualizing (i) intrapineal neurons projecting to the brain in the rainbow trout, (ii) pinealofugal projections to the brain in the rainbow trout and the three-spined stickleback, (iii) retinal ganglion cells in the rainbow trout, and (iv) central pinealopetal projections in the golden hamster. The strength of this method lies in the possibility to perform very accurate applications of HRP to excised brains or brain slices under a stereo microscope, the simple processing of tissue, and the Golgi-like filling of the neuronal profiles. This technique may provide an important complement to currently used HRP-tracing techniques.

Ventrolateral medullary neurons relay cardiovascular inputs to the paraventricular nucleus.

Horseradish peroxidase (HRP) and single-unit recording experiments were done in cats to identify neurons in ventrolateral medulla (VLM) that project directly to the paraventricular nucleus (PVH) and relay cardiovascular information from carotid sinus (CSN) and aortic depressor (ADN) nerves. After diffusion of HRP into the PVH, retrogradely labeled neurons were observed in the VLM. The region of the VLM containing HRP-labeled neurons was then explored for single units antidromically activated by stimulation of the PVH in chloralosed, paralyzed, and artificially ventilated cats. These units were then tested for their responses to stimulation of the CSN and ADN. Antidromic potentials were recorded from 100 units in the VLM. Of these units, 65% were orthodromically excited by stimulation of buffer nerves; 28 by only CSN, 19 by only ADN, and 18 by both CSN and ADN. The axons of antidromically activated units responding to buffer nerves conducted at slower velocities than those of nonresponsive units. These data demonstrate that VLM neurons projecting directly to PVH integrate cardiovascular afferent information and suggest that these VLM neurons may be involved in the control of the activity of magnocellular neurosecretory neurons in the PVH during activation of baroreceptor and chemoreceptor afferent fibers.

The distribution of centrifugal terminals in the pigeon retina.

The terminals of the centrifugal fibres of the pigeon retina have been labelled by anterograde axonal transport or diffusion of horseradish peroxidase, and their distribution studied in whole amounts of the retina. Centrifugal terminal arborisations are concentrated in a band near the projection of the horizontal meridian on the retina. Within this band there are high density areas next to the area centralis and in the mid-temporal retina. The arborisations are largely absent from the red field and are nearly randomly arrayed. Their distribution resembles that of the displaced ganglion cells but not of other retinal neurons. We estimate that approximately 7,000 centrifugal terminal arborisations are present in each retina. Approximately two-thirds of these are convergent, with branches ending in large terminals clustered around a single cell in the amacrine sublayer; often penetrating up to 10 microns into the inner nuclear layer. The remainder are divergent endings which innervate a larger area of retina with widely spaced small terminals about 1 micron in diameter. Possible synaptic contacts between centrifugal terminals and displaced ganglion cells are occasionally seen; these contacts and the similarity in distribution suggest a link between the centrifugal and accessory optic systems.

Type II cochlear ganglion cells in the chinchilla

In order to ascertain whether Type II cochlear ganglion cells project to the brain, we have studied the retrograde transport of horseradish peroxidase (HRP) from the cochlear nucleus to the spiral ganglion of the chinchilla. In this animal there exist two types of ganglion neurons, which closely correspond to those previously described in guinea pigs, cats and rats. As in the guinea pig, the majority population (Type I) consists of relatively large, myelinated neurons. The minority population (Type II, 10% of the total population) consists of small, mostly unmyelinated cells, with filamentous cytoplasm and finely grained nuclear chromatin. Type II neurons tend to be clustered toward the peripheral side of Rosenthal's canal, often in close proximity to the intraganglionic spiral bundle. By 24 h after injections of HRP into the cochlear nucleus, incubation of the cochlear ganglion in diaminobenzidine/H2O2 reveals abundant HRP label in both Type I and Type II neurons. Type II neurons, however, tend to be labelled less intensely than Type I neurons. Control experiments, consisting of spillage of HRP solution over the cochlear nucleus, were carried out to determine how much HRP might be picked up by neurons after HRP diffusion. Comparison of cochleae from injected animals and from the control animals suggests that most of the label that was found in ganglion neurons after cochlear nucleus injections represents axonally transported HRP. We conclude, at least tentatively, that Type II neurons project to the brain. The fact that less label is found in Type II neurons that in Type I neurons suggests that the former have thinner axons and/or finer terminals in the cochlear nucleus.

Morphological observations on superficial medullary CO 2 — Chemosensitive areas

Physiological investigations have indicated that the ventrolateral surface of the medulla oblongata is involved in the chemical drive to respiration. In this investigation, light and electron microscopic investigations of the 3 chemosensitive regions reveal the following. (1) Evaginations of the ventral surface abut the overlying pia mater thereby delimiting discrete compartments; invaginations of the surface delimit wide cisternae lined with basement membrane. Neuronal elements with numerous synapses. were found scattered among astrocytic processes of the marginal glia in intermediate and caudal chemosensitive areas Microvasculature are conspicuously absent from the marginal glia. Intramedullary vessels are surrounded by perivascular spaces and the endothelium shows zonulae occludentes at cell junctions. (2) Horseradish peroxidase (HRP) applied to the ventral surface diffused throughout the interstitial and perivascular compartments, into synaptic clefts and neuronal soma. Diffusion of HRP into blood vessels was blocked at zonulae occludentes. Following intravenous of HRP, no reaction product was found outside cerebral vasculature in chemosensitive areas. (3) In spontaneously breathing cats, 2% procaine applied to the caudal chemosensitive area resulted in respiratory depression which began with the second breath. It is proposed, that substances which stimulate or depress respiration, when applied to the ventral medullary surface, produce their effects on superficial neurons located in the intermediate and caudal chemosensitive areas after diffusion through interstitial spaces.

Postnatal histogenetic death of rat forelimb motoneurons

Previous workers have reported a five fold decrease in motoneuron numbers occurring from birth to adulthood in the rat. It has also been reported that forelimb muscles receive connections from motoneurons in both ventral horns prior to day 14 of life in the rat and that the contralateral cells subsequently degenerate completely by day 21. In the present study, postnatal changes in motoneuron number and distribution within the ventral horn were studied in the rat using the technique of retrograde horseradish peroxidase (HRP) transport following intramuscular infusion of HRP into biceps brachii (BB). Peripheral nerves other than those to BB were sectioned and ligated to control for HRP diffusion. The spatial organization of BB motoneurons was compared in animals 9-14 days old and adult animals using computer 3-D reconstruction. This allowed observations of the BB motoneurons from a variety of perspectives in relationship to a number of spinal cord landmarks and avoided the necessity for transverse and horizontal sectioning of the cords in alternate animals. A decrease of 50-75% in the number of HRP filled motoneurons number was found from birth to adulthood (adults 98-150, neonates 172-243). The spatial arrangement of the BB motoneurons with regard to the root entry zones and other landmarks such as the dorsolateral convexity of the ventral horn, remains constant from birth to adulthood. No evidence was found to support the existence of inappropriate connections from motoneurons in either the ipsi or contralateral ventral horns of neonate rats. It is suggested that the comparatively small decreases in motoneurons postnatally could be a continuation of the histogenetic processes of cell death begun in utero and is related to the postnatal development of the central and peripheral connections of motoneurons.

Cells of origin of propriospinal connections to cat lumbosacral gray as determined with horseradish peroxidase

Propriospinal cells projecting to the lumbosacral spinal cord of cat were identified using the technique of retrograde transport of horseradish peroxidase (HRP). An injection technique was used to preferentially label cells via their terminals rather than through damaged axons of passage. Large volumes (1 to 5 μl) of 35% HRP were slowly ejected from a micropipet penetrating the cord dorsum midway between the dorsal root entry zone and the midline, while the pipet was slowly withdrawn from a depth of 1500 to 2000 μm below the cord surface. This technique resulted in diffusion of HRP throughout the gray matter on the side of the injection and, usually, sperad to the contralateral gray matter. HRP-labeled cells were observed from C1 to S3 after injections in segments L3-S3. Significant differences were seen in the rostrocaudal distributions of labeled cells after injections at different lumbosacral levels: specifically, L6-L7 injections resulted in less labeling of cells at cervical and high thoracic levels than injections that included segments L3-L5 or S1-S3.

Characteristics of capillary permeability in nasal mucosa.

The transendothelial passage of horseradish peroxidase (HRP) and colloidal carbon and diffusion of HRP in pericapillary space, injected intravenously into rats, was studied at the light microscopic and ultrastructural level in the nasal mucosa. In the lamina propria of the mucosa in the middle third of the nasal septum of rats, the capillaries directly beneath the epithelium were mostly with fenestrae, while the capillaries around the nasal gland were without the fenestrae. The permeability of the capillaries in the lamina propria of the nasal mucosa was very high and some of the endothelial cells were wide open, like liver sinusoid, allowing free passage of carbon pasrticles. Marked transendothelial passage was also noted at ten seconds after HRP infusion through the capillaries without fenestrae located around the nasal gland. On the other hand, no extracapillary leakage of HRP was noted even at 150 seconds after its infusion in the capillaries without fenestrae located in the muscles. The permeability of small venules located in the lamina propria of nasal mucosa also was extremely high. The reasons for the greater permeation of HRP through capillaries of nasal mucosa are as follows. First, the endothelial cell junction was loose in the nasal mucosa and in some capillaries endothelium had holes like liver sinusoid. Second, interstitium surrounding the capillary was loose in nasal mucosa.

Ascending and descending components of the medial forebrain bundle in the rat as demonstrated by the horseradish peroxidase-blue reaction. I. Forebrain and upper brain stem.

The ascending and descending components of the medial forebrain bundle (MFB) were investigated by means of horseradish peroxidase (HRP) with a sensitive substrate. The HRP was injected iontophoretically into the MFB at various levels from the anterior commissure to the posterior hypothalamus. In order to prevent the diffusion of HRP to other brain areas, a double micropipette system was used. The descending components of the MFB are derived from (1) the anterior cingulate area, infra- or prelimbic area, and sulcal cortex, (2) the lateral septal nucleus and diagonal band, (3) the bed nucleus of the stria terminalis, (4) the paraventricular nucleus (5) the substantia innominata, (6) the amygdaloid complex (AM), (7) the ventromedial (VM) and dorsomedial (DM) hypothalamic nuclei, (8) the entopeduncular nucleus and (9) nucleus periventricularis stellatocellularis. The ascending components of the MFB originate in: (1) the medial preoptic nucleus, (2) the nucleus periventricularis stellatocellularis and rotundocellularis, (3) the posterior hypothalamic nucleus, (4) the parafascicular nucleus, (5) the ventral premammillary nucleus, (6) the substantia grisea periventricularis, (7) the lateral habenular nucleus, (8) the VM and DM, (9) the paratenial nucleus, (10) the AM and (11) the arcuate nucleus.

Early development and migration of the trigeminal motor nucleus in the chick embryo.

The development of the trigeminal motor nucleus in the chick embryo was studied using autoradiographic, cell staining, fiber staining, and axonal transport techniques. It was found that this nucleus arises very early in neurogenesis, with the first cells produced at 48 hours of incubation (stage 12), peak cell production at 50--56 hours (stage 15), and neuroblast proliferation completed by 72 hours (stage 18). As has been described in mammalian embryos, the primordial trigeminal cells move from the ventricular layer to accumulate as part of the common medial column, and later migrate in a ventrolateral direction to form the definitive lateral motor nucleus. The first identifiable component of the trigeminal system is the semilunar ganglion, which flanks the neural tube at stage 12, and sends afferents into the metencephalon by stage 13. By stage 12-13, the medial column cells are first apparent, and at stage 14, a few of these medial column cells have moved to begin formation of a lateral nucleus. At this time, a thin motor root can be seen exiting the brainstem. During subsequent stages, migratory traffic from medial to lateral regions increases, with cells frequently moving in association with fiber processes in the marginal zone. These fibers are presumed to emanate from secondary sensory, reticular, and medial column neuroblasts. By day 5, the medial column is greatly depleted and by day 6--7, the definitive lateral motor nucleus is formed. Beginning at 5 days, the dorsal motor nucleus can be detected, with cells from the lateral nucleus appearing to stream in a dorsomedial direction for its formation. Injections of horseradish peroxidase (HRP) into the mandibular process of the first visceral arch resulted in retrograde labeling of lateral nucleus cells as early as 3.5 days of incubation. In addition, migrating cells, intermediate between medial column and lateral nucleus, were similarly labeled. These observations indicate that processes of the lateral nucleus cells and those of migrating cells are well into their peripheral field at this age, but we cannot conclude that neuromuscular affiliations have been established, due to the possibility of HRP diffusion and growth cone uptake.

Observations of HRP labeling following injection through a chronically implanted cannula — A method to avoid diffusion of HRP into injured fibers

One of the limitations of the horseradish peroxidase (HRP) tracer method is the diffusion of HRP into injured axons resulting in unintended labeling of neurons not terminating in the injection area. To overcome this limitation, an experiment was designed to inject the HRP through an implanted cannula after degeneration and healing had taken place. It was shown that implantation of a cannula into the internal capsule significantly decreased the number of labeled axons in the injection site, thus limiting the unintended labeling of neurons from that injection. When injections followed implantation of the cannula by 24 h or more, fibers damaged by the cannula had healed or degenerated sufficiently that intraaxonal diffusion of HRP into those injured fibers did not occur. A significant difference between control (without the cannula) and experimental (with the cannula) injections was observed. Extensive axonal and neuronal labeling following the control injections was seen at the injection site and caudate nucleus, and in the thalamus and parietal cortex, respectively. Experimental injections resulted in sparse axonal and neuronal labeling evident mostly with the larger injections of HRP.