INTRODUCTION

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Sneezing, itching, and watery secretion, the outstanding symptoms of rhinitis, are a consequence of changes in blood flow, vascular permeability, and glandular secretion mediated by the release of various bioactive molecules (1). Moreover, recent observations in humans, as well as experimental work in animal models, have emphasized the activation of local nerve reflexes as one of the main factors in the pathogenetic pathways of rhinitis (2, 3).

The vascular supply of the nose (4) consists of resistance (arteries, arterioles), capacitance (veins, venules, sinusoids), and exchange vessels (capillaries). Arteries supplying the mucosa arise from the perichondrial and periosteal arteries, and give branches to the capacitance vessels before forming a subepithelial network of fenestrated capillaries (5). Blood flow into the sinusoids regulates nasal patency and airflow (4) and is influenced by emotional stress, temperature, hormones, and several bioactive molecules (5). Parasympathetic nerves are vasodilator, sympathetic nerves are vasoconstrictor, and sensory nerves are able to release dilator neuropeptides; besides, most inflammatory and immunologic mediators are vasodilator (6).

Acetylcholine, catecholamines, various peptides and also nitric oxide participate in nasal vascular control (3). These bioactive molecules could arise from both sensitive and autonomic nerve fibers and from neuroendocrine cells widely dispersed in the nasal mucosa (7). The adult human nasal mucosa exhibits dense nerve networks containing vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), or its C-terminal peptide (CPON), substance P (SP), calcitonin gene-related peptide (CGRP) among others (7). Sympathetic fibers carry both norepinephrine and NPY (8). Immunoreactivities for NPY and CPON show full colocalization and are mainly found in perivascular fibers (7). The subepithelial region contains a dense plexus of SP- and CGRP-immunoreactive fibers, but these nerves also appear around blood vessels (7, 9). However, the role of different nerve classes in the pathogenesis of allergic rhinitis is still unknown.

A key issue for the management of chronic rhinitis is to understand the factors generating the unceasing mucosal hyperreactivity, a condition where even a minimal stimulus will generate a symptomatic cascade. Changes in vascular innervation could be one of the factors involved in the maintenance of rhinitis. Therefore, we have analyzed the innervation of nasal blood vessels in children with and without symptoms of chronic rhinitis. The general distribution of nerves was studied using the antiserum against ubiquitin carboxyl-terminal hydrolase or protein gene product 9.5 (PGP), a general neuronal marker (10) reflecting the distribution of all kinds of nerves. Particular classes of nerves were studied using antisera against CPON, a neuropeptide mainly found in sympathetic fibers, and against CGRP, a characteristic peptide of sensory fibers (11).

	METHODS

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Patients

Biopsies from nasal mucosa were obtained from children 4 to 9 yr of age attending the otorhinolaryngology department of Hospital Escuela José de San Martín. Surgical procedures were done under a protocol approved by the corresponding Teaching and Research Board. A prior indication for amygdalectomy was a mandatory requisite for entering this study since this indication also involved an indication for general anesthesia, thus avoiding a biopsy under local anesthesia in these young patients. All parents were informed about the goals and risks of the procedure, and they gave their written consent.

The control group was formed by six children without a history of persistent nasal inflammation and free of rhinitic symptoms or signs at the time of surgery. The test group constituted five other children with persistent symptoms and signs compatible with chronic inflammation of the nasal mucosa: nasal obstruction, persistent watery secretion, sneezes, direct visualization of vascular engorgement, and edema of the mucosa. All these children had an elevated plasmatic IgE and had received multiple local and general treatments during the previous year (vasoconstrictors, antihistaminics, or corticoids) without lasting improvement. They were symptomatic and treatment-free for 1 wk at the time of surgery.

Biopsies

Punch samples from the middle turbinate were obtained with a disposable device (Biopunch, 2.5 mm; Fray Corp., Amherst, NY) and immediately fixed in a mixture of 4% paraformaldehyde and 0.4% picric acid in 0.16 M phosphate buffer solution for 6 to 8 h at room temperature. Specimens were then immersed in 15% sucrose in phosphate-buffered saline (PBS) with 0.01% sodium azide (Merck, Darmstadt, Germany) for at least 24 h. Sections (14 µm) were obtained in a cryostat (Microm; Waldorff, Germany) and mounted on gelatin-coated slides and processed as previously described (12). After delipidizing and blocking endogenous peroxidase with 0.03% hydrogen peroxide, they were incubated with rabbit sera raised against different markers (Table 1) for 48 h at 4° C. Only a small number of markers was used because of the small size of the samples. Slides were then washed in PBS, and sections were incubated successively with biotinylated goat antiserum to rabbit IgG (Vector Laboratories, Burlingame, CA) and freshly prepared ABC (Vectastain Elite; Vector). Peroxidase activity was revealed using diaminobenzidine with nickel-enhancement. Control slides were incubated with preimmune serum or omitting the primary or secondary antibodies. Vascular innervation was analyzed using arbitrary scales (see RESULTS), which were compared with the Kruskall-Wallis test for nonparametic data (13).

Image Analysis

Microscopic images for quantification were obtained with a Zeiss photomicroscope fitted with a black and white CCD camera (COHU Inc., San Diego, CA). They were digitalized with an Oculus-TCX True Color Frame-Grabber (Coreco, V. St. Laurent, PQ, Canada). Bioscan Optimas, version 4.10 was used for analysis of vascular parameters. Complete transverse sections of vessels showing all the histologic layers were selected for measurement. Tangential sections were excluded from the quantitative analysis. Thickness of vascular walls was measured at four sites located on the intersection of each vessel with the vertical and horizontal axes of the screen. The average of the four measures was taken as an indication of wall thickness (WT). The field of each vessel was defined as the area enclosed by the outer aspect of the perivascular nerve cuff. The proportion of immunostained versus nonimmunostained areas of the vessel wall was measured by segmentation of the field according to three different grey level thresholds: lumen (no staining), total wall (all grey level values), and innervated wall (high grey level values). The ratio between innervated wall and total wall was taken as an index of the innervation density (ID) of the blood vessel since this parameter (which should not be modified by slight slanting of the vascular profiles) allowed the comparison of vessels with different diameters. Data were compared using multiple analysis of variance (MANOVA) (13). The researcher in charge of image evaluation and quantification was blind to the rhinitic or nonrhinitic status of each patient.

	RESULTS

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Biopsies of turbinate mucosa from nonrhinitic patients contained several types of blood vessels. Medium-size arteries and small arterioles had a thick coat of circumferentially oriented muscle cells. Medium-size and small venules exhibited a thin muscular coat and a wide lumen. Arteriovenous anastomoses (AVAs), the characteristic vessels of the turbinate mucosa, showed a thick muscle coat with an epithelioid appearance where smooth muscle cells ran through different axes. Their intima was devoid of an inner elastic membrane. Sinusoids, occupying the deeper regions of the specimen, were not present in all the samples.

As demonstrated by PGP immunoreactivity, each kind of vessel displayed a characteristic innervation pattern. A compact cuff of fibers surrounded arterial muscle layers (Figures 1 to 4), whereas a much smaller number of nerve fibers associated with venous profiles. Sinusoids showed few, if any, surrounding nerve fibers. Arteriovenous anastomoses contained a moderate amount of nerves; however, these fibers penetrated deeply within the muscle coat, sometimes approaching the lumen (Figures 5 to 9). All these blood vessels also exhibited CPON-immunoreactive fibers. A much smaller number of vascular fibers was labeled by the antiserum against CGRP, around both arteries and AVAs (Figures 2 to 4 and 6 to 8).

Vascular structure in rhinitic samples followed the same pattern (Figures 9 to 13). However, contorted AVA profiles with smooth muscle cells running in random directions were most often found in the rhinitic biopsies. Two different procedures were used to compare vascular innervation in each group of patients. First, the innervation pattern of arteries and AVAs was classified in arbitrary grades (Figures 2 to 4 and 6 to 8). Second, PGP perivascular immunoreactivity was quantified with an image analysis protocol program.

For arterial innervation, the following grades were defined: 0, no innervation detected; 1, nerves formed an incomplete cuff; 2, nerves formed a complete cuff around the artery; 3, the nerve cuff was very thick. The grade assigned to each sample reflected the innervation density of the most richly innervated arteries from that sample. All samples contained a large amount of PGP-immunoreactive fibers since this is a general neuronal marker staining all classes of nerves. CPON immunoreactivity was also present in a large proportion of the periarterial fibers, whereas CGRP immunostained fibers were rather scanty (Figure 14).

View larger version (17K): [in this window] [in a new window]   Figure 14.   Bar charts representing the average innervation grade (± standard error) for PGP, CPON, and CGRP in arteries (top panel ) and AVAs (bottom panel ) from rhinitic and nonrhinitic samples. Notice the higher grades of rhinitic patients. After correction for ties, analysis with the Kruskal-Wallis test indicated that there were significant differences in the medians (p < 0.004 for arterial innervation, and p < 0.001 for AVAs).

The innervation pattern of AVAs was classified into the following grades: 0, no innervation detected; 1, few perivascular nerves with nerve-free regions larger or more frequent than innervated regions; 2, numerous perivascular and intramural nerves; 3, nerves formed a complete perivascular cuff and numerous fibers approximated to the endothelium. Bar charts reflecting the average grades for each neuronal marker in the different samples indicated that both arteries and AVAs exhibited a richer innervation in rhinitic than in nonrhinitic patients (Figure 14). Expansion of the perivascular fibers was most noticeable in samples immunostained for PGP or CPON. Observations suggested that CPON immunoreactivity was present in most arterial nerves (Figures 10 and 11). In AVAs, by contrast, CPON-immunoreactive fibers represented just a small fraction of all perivascular fibers immunostained by PGP (Figures 12 and 13). No changes of CGRP-immunoreactive fibers could be detected.

Quantitative differences of vascular innervation could be demonstrated by image analysis of perivascular PGP immunoreactivity (Table 2 and Figure 15). In the nonrhinitic mucosa, measurements of PGP-immunoreactive areas indicated that nerve fibers occupied a significant proportion of the arterial wall, accounting on average for about 10% of its total area. Immunostained nerves occupied a larger proportion of the arterial wall in those vessels with a thinner wall. Thus, in arteries with a wall thickness less than 25 µm, as much as 30 to 40% of the wall area was occupied by PGP immunoreactivity. No significant differences were detected between the wall thickness of rhinitic and that of nonrhinitic patients. However, arteries from rhinitic patients contained a larger proportion of PGP-immunoreactive fibers than did those from nonrhinitic patients (Table 2). The increase in innervation density was much more evident in those with the largest wall thickness (Figure 15), and, as indicated by MANOVA, it was significantly different (p  0.001).

View larger version (17K): [in this window] [in a new window]   Figure 15.   Innervation density (ID) of arteries and AVAs, represented by the proportion of vascular wall immunostained for the general neuronal marker PGP, is plotted against the wall thickness of each vascular profile. (Upper panels) The distributions of arterial IDs from nonrhinitic and rhinitic patients are markedly different, being significantly larger in the rhinitic patients. (Lower panels) AVAs from nonrhinitic patients also exhibit a much lower ID than AVAs from rhinitic patients.

Quantitative analysis of PGP-immunoreactive fibers surrounding AVAs gave similar results (Table 2 and Figure 15). In both nonrhinitic and rhinitic patients there was an inverse relationship between wall thickness of the vessel and its innervation density. No significant differences could be detected between the AVA thickness of each group. By contrast, in the rhinitic patients there was a significant increase of PGP immunoreactivity within the AVA walls (p  0.001), almost duplicating the innervation densities measured in nonrhinitic patients.

A comparison between the epithelial innervation of rhinitic and nonrhinitic mucosas could not be made since the epithelium was not preserved in nonrhinitic specimens. In the subepithelial region, numerous fibers were immunostained with the PGP, CGRP, and SP antisera. When the epithelium was present, it could be observed that these fibers crossed the basal membrane forming intraepithelial ramifications.

Neuroendocrine cells were immunostained with the CGRP, CPON, and PGP antisera. They were randomly distributed in the subepithelial tissue or between glandular acini. No qualitative differences were discerned between rhinitic and nonrhinitic patients, and a quantitative study was hindered by their irregular distribution.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Recent advances in the mechanisms of mucosal inflammation emphasize the role of various interactions between neural mediators, vascular wall structures, and cells of the immune system (3, 14). In children, the problem of chronic rhinitis is marked by two main questions: whether its etiopathogeny and treatment differ from those of adult rhinitis, and whether this pediatric condition is related to other respiratory diseases in the adult. It is expected that our findings about the vascular innervation pattern in children nasal mucosa and its modifications under rhinitic conditions should be helpful in understanding these issues.

The vascular innervation pattern in nonrhinitic children followed the same general plan described in adults (9). Nerves were found mainly around arteries, but they were also present around AVAs and veins. The high innervation density of nasal arteries in comparison with other nasal blood vessels has already been reported for NPY and VIP immunoreactivities (7), but perivascular CGRP-immunoreactive fibers were not previously described. Few nerve fibers appeared around veins, and we did not detect them around sinusoid capillaries, as described in the adult mucosa (7). In the young turbinate, immunostaining for PGP and CPON clearly demonstrated nerve fibers within the smooth muscle layers of the AVA tunica media. However, according to ultrastructural studies in the adult nasal septum (15) nerve fibers would be present only in the adventitia of these vessels and would never penetrate their tunica media. A similar adventitial distribution has been described in the skin (16). Both AVAs and venous sinusoids are scarcely developed in human fetal nasal tissues (17) and probably have a protracted postnatal maturation; therefore, these differences in their innervation pattern could represent age-related phenomena that might be involved in the inflammatory responses of children's nasal mucosa.

Vessels from rhinitic patients were much more densely innervated than those from nonrhinitic children, the differences being demonstrated both by qualitative and quantitative methods in arteries and AVAs. Innervation density (ID), as demonstrated by PGP immunoreactivity, was almost doubled in rhinitic arteries, the increase being most noticeable in the largest ones. However, no differences in the thickness of vascular walls could be detected.

Peripheral nerves are probably much more reactive than is usually thought since an increase in the number and intensity of nasal SP- and CGRP-immunoreactive fibers has been reported after only 10 min of intranasal application of toluene diisocyanate in guinea pigs previously sensitized to this agent (18). These fibers return to control levels within 7 d of the challenge. Because at the same time there is a decrease of SP- and CGRP-immunoreactivities in the trigeminal cell bodies, these changes indicate a displacement of bioactive molecule from the soma to the terminals. By contrast, the increase of PGP immunoreactivity around human rhinitic vessels could represent a more permanent increase of nerve terminals since ubiquitin-associated pathways are related to degradation of neurofilaments in situ (19), and PGP is transported in the axon exclusively with a slow component (20).

Blood vessels of the nasal mucosa are richly innervated by sympathetic fibers releasing noradrenaline, neuropeptide Y, and somatostatin (8). In both rhinitic and nonrhinitic biopsies, the vast majority of CPON-immunoreactive nerves innervated arteries and arterioles. Comparison with PGP immunostaining indicated that CPON-immunoreactive fibers were the most abundant nerves associated with those vessels.

In both rhinitic and nonrhinitic mucosas, AVAs exhibited a characteristic innervation pattern that was different from the arterial one. These vessels had abundant CPON-immunoreactive fibers, but less numerous than those immunostained by PGP. This probably reflects the different responses of resistance and capacitance vessels to sympathetic stimulation (5, 21). In the nasal mucosa of cats, about 60% of the blood flow is normally shunted through AVAs (22). The larger proportion of CPON-immunoreactive nerves in arteries compared with AVAs bears with pharmacological experiments demonstrating that vascular resistance and vascular volume can be separately influenced by nerves and mediators (23).

Innervation of nasal AVAs probably includes other types of fibers, including cholinergic, VIP-, CGRP-, SP- and nitric-oxide-synthase-containing nerves, as has been shown in several locations (24, 25). Nasal ateriovenous flow is less sensitive to ADP than nasal capillary flow (26), suggesting that purinergic nerves could be differentially distributed to each type of vessel.

Vascular hyperinnervation, together with observations about the shape and relative abundance of AVA profiles in the rhinitic mucosa, suggest that chronic mucosal inflammation would be accompanied by an abnormal program of vascular maturation. Arterial hyperinnervation in the rhinitic mucosa resembles a similar phenomenon described in children with pulmonary hypertension, where arteries of the lung respiratory unit are prematurely innervated by sympathetic fibers, and vascular muscle cells apparently undergo an accelerated maturation (27). Changes in the onset of sympathetic innervation could induce modifications in the response of adrenergic and cholinergic receptors, as shown by the effects of chronic NPY on heart muscle (28). Moreover, NPY can stimulate growth of vascular smooth muscle cells (29).

If the observed increase in nerve immunoreactivity actually represented an increased neurotransmitter release by periarterial nerves, the rhinitic mucosa could resemble the condition occurring in rhinitis medicamentosa associated with abuse of local -adrenergic agonists, where the strong nasal obstruction is no longer responsive to vasoconstrictor drugs (21). Increase of neuromediators released in the perivascular environment could also be involved in modulation of immune and inflammatory phenomena. Thus, NPY and SP release histamine from mast cells (30) and, together with endothelin, can modulate several polymorphonuclear functions (31).

In our samples, the epithelium was well preserved in rhinitic patients but not in the nonrhinitic ones. Although it has been generally thought that airway inflammation includes grossly denuded airways, or at least a severely disrupted epithelium, this concept has been recently challenged since allergic airway disease may be associated with decreased mucosal absorption (32). Our observations are in line with this conservation of the mucosal barrier function in chronic rhinitis.

It has been speculated that upper airway disease in children could be associated with bronchial hyperreactivity and asthmatic disorders in the adult (33, 34). Hyperinnervation of the rhinitic mucosa could be the basis not only for the maintenance of the rhinitic state but also for disturbances of nasobronchial and sinubronchial reflexes presumably involved in the genesis of lower airway hyperreactivity.