The Histochemical Journal31: 153–160, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

Effect of inflammatory stimuli on the silver staining pattern of therat carotid endothelium

Marıa Gabaldon & Ana MarquesCentro de Investigaci´on, Hospital La Fe, Unidad Histoquimia, Avenida Campanar 21, 46009 Valencia, Spain

Received 5 May 1998 and in revised form 1 December 1998

Summary

Silver nitrate stains the intercellular junctions of the endothelium and other cytoplasmic or membrane components. Two proto-cols are described for the silver staining of rat carotid endothelium that exclude the use of pressurized fixatives and simplify thetechnique previously described for rat aorta. The entire surface of the carotid endothelium was examined and several parameters(stigmata, granularity, clustering of anionic sites, transversal lines, weakening of silver lines and leukocyte adhesion) wereevaluated. We studied the pattern of silver staining in two situations: (1) endothelial activation and (2) neurogenic inflammation.Endothelial activation was produced by the intravenous administration of a proinflammatory albumin or polyinosinic acid. Bothproducts cause a marked increase in leukocyte adhesion concomitant with a decrease in argyrophilia and a weakness or lossof silver lines. Neurogenic inflammation, which is mediated by substances released from sensory nerves, was induced by theintravenous administration of substance P or capsaicin. Both stimuli produced an increase in argyrophilia and weakness or lossof silver lines. Substance P caused a clustering of anionic sites, whereas this phenomenon was more discrete with capsaicin.Nearly 80% of all examined rats (controls and inflammatory stimuli treated) showed endothelial membrane disruptions formedby clusters of cells often in the shape of streaks aligned with the long axis of the vessel. The detection of these discontinuitiesis important, as loss of endothelial integrity is central in the initiation of pathological events.

Introduction

The staining of the interendothelial junctions by silver nitrateis a useful technique for determining the size, shape andintegrity of endothelial cells. In a previous paper (Gabaldon1987), methodological aspects related to the silver stainingof the endothelium of rat aorta were reported. Conditionsthat were optimal for aorta gave inappropriate silver patternsin carotid artery, as the silver lines were either too weak orabsent. On the other hand, the method used to date (Gabaldon1987) required the handling of pressurized fixatives to achievevessel fixation at physiological pressure, and this posed a riskfor untrained personnel. We have modified the previous tech-nique with three goals in mind: (1) to improve labour safetyexcluding the use of pressurized fixatives; (2) to simplify theprocess by eliminating intermediate steps, and (3) to establishnew conditions for the silver staining of endothelium of ratcarotid.

Silver staining is an extraordinary selective technique,which apart from the interendothelial junctions, also stainsother structures of unknown nature. From the literature accu-mulated over the years, it seems evident that lesive stimulimodify the pattern of silver staining. Argyrophilic cells in theaortic endothelium have been described after administrationof hypercholesterolemic diets (Zaikinaet al. 1982, Spanet al.1992), in alloxan induced diabetes (Dolgovet al. 1982), andin carotid artery after transmural electric stimulation (Kling

et al. 1987). Infections byMycoplasma pulmonis, Kilhamvirus (Gabaldonet al. 1992) and cytomegalovirus (Spanet al.1992) also increase the argyrophilia of the rat aortic endothe-lium. Some of these stimuli may produce an inflammatoryresponse in the endothelium, and this phenomenon, knownas endothelial activation, is manifested by several phenotypicchanges – one of the most important being the expressionof receptors involved in leukocyte adhesion. To determinewhether endothelial activation can modify the pattern ofsilver staining, we have utilized two products which afterparental administration markedly increase leukocyte adhe-sion to rat carotid endothelium: a bovine albumin obtainedby heat shock and polyinosinic acid (Zuniga et al. 1997).Apart from the proinflammatory activity we have observedin vivo, this latter product has shown the same activityin vitroby producing an important expression of ICAM-1 receptorsin cultured endothelial cells (Palkamaet al. 1993).

To determine whether nervous stimulation can modify thepattern of silver staining we have induced a type of inflam-mation mediated by substances released from sensory nerves(neurogenic inflammation). McDonald (1994) showed thatthe intravenous administration of substance P produced inthe endothelium of postcapillary venules an increase in: (1)vascular permeability – the gaps made visible by silver stain-ing as black dots along the endothelial cell border – and (2)the adhesion of leukocytes, frequently surrounded by silverrings and located at the intercellular junctions – the sites of

154 M. Gabaldon & A. Marques

leukocyte adhesion being different to the endothelial gaps. Inorder to determine whether neurogenic inflammation modi-fies the pattern of silver staining in carotid in the same wayas in postcapillary venules, we used substance P – asensorynerve mediator – and capsaicin, a drug that releases endoge-nous neuropeptides such as substance P from sensory nerves,and produces an increase in leukocyte adhesion (Umenoet al.1990) and vascular permeability (Piedimonteet al. 1990).

Materials and methods

Products

Bovine albumin (A-7906), polyinosinic acid (5′), substanceP (S-6883), capsaicin (M-2028), paraformaldehyde (P6148)and gum arabic were obtained from Sigma Chemical Co.Silver nitrate and 25% glutaraldehyde were provided byMerck & Co.

Animals

Specific pathogen-free male Wistar rats weighing 200–250 gwere used (B & K, Universal Ltd., England). Animals wereanesthetized with 50 mg/kg sodium pentobarbital i.p. Albu-min was dissolved in saline and administered at 25 mg/100 gas a bolus (0.25 ml/100 g). Poly I was dissolved in saline andadministered at 800µg/100 g as a continuous infusion for 1 h(0.5 ml/100 g/h). A stock solution (×10) of substance P wasprepared at 50µg/ml in 5 mM acetic acid–0.85% NaCl, andaliquots were frozen. Upon use, solutions were diluted withthe vehicle. Substance P was administered at 5µg/kg (0.1ml/100 g) during 20 sec. A stock solution of capsaicin (×20)was prepared at 1.75 mg/ml in 10% ethanol–5% Tween 80–0.76% NaCl, and aliquots were frozen. Capsaicin was firstdissolved in ethanol before adding the other components.Upon use, solutions were diluted with saline. Capsaicin wasadministered at 175µg/kg (0.2 ml/100 g) during 2 min. Allsolutions were administered into the saphenous vein.

Silver staining

Two protocols have been utilized differing in access route,perfusion flow, AgNO3 concentration and perfusion time, andbromides perfusion time. Both protocols have the following incommon: (1) fixative, 1% paraformaldehyde–2% glutaralde-hyde in 0.1 M phosphate buffer pH 7.4; (2) washing solution,8.9% saccharose in HEPES buffer pH 7.4; (3) bromides, 0.1%NH4Br–0.3% CoBr2 ·H2O; (4) drainage of perfusion throughthe external jugular veins, and (5) illumination under a desklamp with a 60 W bulb at distance of 20 cm, for 30 min.

Protocol 1A 20 G Vasocan cannula was inserted in the abdominal aortaand solutions were applied as follows: (1) fixative at 25

ml/min, for 4 min. Fixative was introduced manually withtwo 50-ml syringes, at a high flow and near-physiologicalpressure, for quick blood elimination; (2) fixative at 5 ml/minfor 6 min, using an infusion pump; (3) washing solution with40 mM HEPES buffer; (4) 0.07% AgNO3 in washing solution;(5) washing solution; (6) bromides; (7) washing solution; (8)fixative, and (9) water. Starting from step 3, perfusion wasperformed manually with syringes at 5 ml/min for 2 min.

Protocol 2A 22 G Vasocan cannula was inserted in the right carotid andsolutions were applied as follows: (1) fixative at 25 ml/min,for 4 min; (2) fixative at 8 ml/min, for 6 min (infusion pump);(3) washing solution with 20 mM HEPES buffer; (4) 0.05%AgNO3 in washing solution; (5) washing solution; (6) bro-mides; (7) washing solution; (8) fixative, and (9) water. Start-ing from step 3, perfusion was performed manually withsyringes at 13 ml/min for 1.5 min.

Tissue preparation

The left common carotid was removed by sharp dissection.The carotid was cut beyond root level at both ends. To per-form posterior staining, the carotid must be joined to the aorticarch caudal and must carry short segments of both the externaland internal carotids cephalad. Carotid was cleaned of peri-adventitial tissue in a petri dish with water and illuminated for30 min. Elimination of this tissue must be complete to avoidan uneven thickness of the preparation, which would causefocusing problems, thereby rendering photomicrography dif-ficult and microscopic observation tiring. A 20 G Vasocancannula was inserted through the aortic arch and minimallyintroduced in the left carotid. A 1-ml syringe filled with Gill’sHaematoxylin No. 3 (formulation in data sheet 192, Poly-sciences Inc., Warrington, PA, USA) was adapted to the can-nula and injected very slowly into the lumen of the carotid,for 75 sec. Syringes were successively applied to the cannulawith: (1) 2 ml H2O; (2) 2 ml of Scott’s tap water substitutefor 5 min (2% MgSO4 · 7H2O–0.2% NaHCO3), and (3) 2 mlwater. The purpose of applying solutions through the lumenand of maintaining the carotid bifurcation is to avoid con-tact of hematoxylin with the adventitia and restrict stainingto the endothelium. The profuse staining of smooth musclecells through the adventitia produces a blurry field that makesmicroscopic observation difficult. Carotid was cut at the levelof the aortic arch and bifurcation and slit open longitudi-nally. Whole mounts of carotid were obtained as describedpreviously and mounted (Gabaldon et al. 1994). In order toachieve better uniformity in thickness, a weight of approxi-mately 400 g (5×1.7 cm) was placed on the mounted prepara-tion until the following day. The aqueous mounting mediumwas prepared as follows: 20 g of gum arabic and 40 mg ofthymol were interposed in a saccharose solution containing40 g saccharose and 40 ml water. The suspension was heatedto 40–50◦C for 30 min with occasional shaking, and aftercooling was centrifuged at 5,500 rpm for 60 min. Foam was

Silver staining of carotid endothelium 155

removed with a vacuum pipette. The solution was decantedand stored in the refrigerator.

Light microscopy

Preparations were examined under×400 magnification bymoving the microscopic field systematically along thecarotid. The continuous scanning provides a complete field ofobservation for detecting any phenomenon regardless of itsfrequency. Evaluated parameters were scored on a semiquan-titative scale from 1 to 3 (1, discrete; 2, abundant; 3, veryabundant). Photomicrography was performed with a BH-2Olympus microscope using a×40 objective, under the fol-lowing conditions: B12 Nikon filter; condenser diaphragm,0.4; light power, 6.5, and Kodak Gold 100 ASA film.

Results

Control rats

Silver staining gives information on the size and shape ofendothelial cells. These parameters vary along the carotidunder hemodynamic influences (Nerem & Girard 1990). Inthe caudal extreme of the carotid, proximal to the aortic arch,cells are narrow, elongated and with interdigitations; silverlines are weak and transverse lines are abundant. This patternis similar to that found in the cranial extreme of the thoracicaorta (data not shown) and possibly represents an adaptationto the hemodynamic stress produced by the non-laminar flowat the curvature and ramifications of the aortic arch. In thecranial extreme of the carotid the pattern is quite different,with wider silver lines, absence of transverse lines and withmore rounded cells without interdigitations. This pattern issimilar to that found in the caudal extreme of the thoracicaorta (data not shown) and possibly represents a response tothe new hemodynamic conditions generated in the proximityof the flow divider.

Silver nitrate stains interendothelial junctions and otherstructures on the cell surface. The parameters we have consid-ered in our evaluation are the following: (1)stigmata, whichare focal silver deposits on or between the cells and gener-ally not exceeding seven per cell; (2) fine granularity, withgrains smaller and much more abundant thanstigmata. Anexcess of fine granularity gives rise to the so-called argy-rophilic cells, which are cells full of silver grains distributedisolatedly or in ribbons along the flow direction, produc-ing a sharp contrast with the clean neighboring cells; (3)gross granularity, with large amorphous silver precipitationon the interendothelial line or on the membrane surface, grossgranularity being frequent in mitotic cells; (4) clustering ofanionic sites, possibly produced by a redistribution of anionicsites along the interendothelial junction, with the disappear-ance of silver lines and the formation of short, intense hyphen-like figures orientated in the direction of flow; (5) transverselines, which are silver lines perpendicular to the direction offlow and much more intense in proximity to the aortic arch

than in the remaining carotid. Transverse lines are associatedwith weak or absent interendothelial junction silver lines andare located at a deeper focal plane than the latter; transverselines represent the cell-to-cell contacts between smooth mus-cle cells of the medial layer (Gottlob & Hoff 1968, 1977);(6) weakness or disappearance of silver lines often accom-panied by a yellowing of the background, and (7) adheredleukocytes. Leukocytes adhered in control rats are normallyfew (Gabaldon et al. 1992) and this low level was scoredas 0. Figure 1(a–f ) shows the staining patterns found in theendothelium of rat carotid. The selectivity of the silver stain-ing allows us to observe a sharp contrast between some cellsand their neighbors (Figure 1(f–h)).

Table 1 shows the mean score of the parameters evaluatedby Protocols 1 and 2. Silver staining is largely influencedby technical factors. Values show a great dispersion due toindividual variations and the use of a short and discontinuousscale. Although normally accepted levels of significance arenot achieved with most of the parameters studied, it seemsclear that the stronger conditions of Protocol 1 (higher AgNO3

concentration, longer action time of AgNO3 and bromidesand less perfusion flow) increases argyrophilia as a whole(stigmata, fine and gross granularity). The weaker conditionsof Protocol 2 decrease argyrophilia and increase the zoneswith weak or absent silver lines. These two protocols seemadequate for staining carotid endothelium with silver nitrateand may represent the two extremes of a range of conditions.The choice of either will depend on the effect produced bythe variable under study. If the variable increases argyrophiliaas a whole, Protocol 1 will be inadequate because exces-sive staining will make it difficult to find differences betweenpreparations. In relation to perfusion flow, we consider theconditions of Protocol 2 to be more adequate because theyare closer to physiological values, thereby precluding arti-facts produced by insufficient distension of the vessel. Whenchanging conditions, the pH of the AgNO3 solution must betaken into account, as silver pattern is affected by this variable(Gabaldon 1987). The pH of the AgNO3 solution was 7.18 inthe two protocols used.

The most surprising finding in thus study was the presencein control rats of plasma membrane disruptions. Woundedendothelial cells were more proximal to the bifurcation thanto the aortic arch, and were found in clusters of various sizesoften in the shape of streaks aligned with the long axis of thevessel. Silver staining detected several patterns: (1) disap-pearance of silver lines, intense transverse lines and grossgranularity, forming short patches of few cells; (2) weakor absent silver lines, gross or fine granularity and rolledendothelial nuclei, forming streaks about 1 mm in length,and (3) holes rimmed by gross granularity with or withouttransverse lines. Seventy-seven percent of the control ratspresented these injury lines, and the number of streaks percarotid was between one to three. Figure 2(a–c) shows someaspects of these plasma membrane disruptions. Plasma mem-brane disruptions of the same characteristics as the controlrats were found in the remaining rats treated with the differ-ent stimuli studied and their vehicles.

156 M. Gabaldon & A. Marques

Figure 1. A–F, Staining patterns in the endothelium of control rats, (A–E, Protocol 1; F, Protocol 2). (A) endothelial pavement without granularity;mitosis with gross granularity (arrow); (B) stigmata; (C) gross granularity. (D) argyrophilic cells; (E) transverse lines (arrow, flow direction) and (F)clustering of anionic sites. F–H, Sharp contrast between some cells and their neighbors, (G–H, Protocol 1). Bar, 20µm.

Silver staining of carotid endothelium 157

Table 1. Mean score of the parameters evaluated in thesilver staining of the carotid endothelium by following Pro-tocols 1 and 2.

Parameter Protocol 1 Protocol 2(5) (8)

Stigmata 1.60± 1.14 0.87± 0.99Fine granularity 2.40± 0.89 1.87± 0.83Gross granularity 2.00± 0.71 0.62± 0.92∗

Clustering of anionic sites 0.20± 0.45 1.25± 1.03Transversal lines 1.20± 0.84 0.37± 0.74Weak or absent lines 1.00± 1.22 1.62± 0.74Leukocytes 0.00± 0.00 0.12± 0.35

∗P < 0.05, Mann–WhitneyU -two tailed test.In parentheses, number of rats in each group. Resultsexpressed as mean± S.D.

Endothelial activation

Table 2 shows the modification of the parameters evaluated bysilver staining of the carotid endothelium, 18 h after admin-istration of two stimuli that cause endothelial activation. Atthis time, proinflammatory albumin and Poly I respectivelyincrease the basal level of leukocyte adhesion×7 and×44(Zuniga et al. 1997). Leukocytes were generally in contactwith the interendothelial junction and were never rimmed bya silver line. None of the two stimuli utilized produced a pref-erential adhesion of leukocytes to any of the staining patternsdescribed previously. Endothelial activation modifies the pat-tern of silver staining by producing a decrease in argyrophiliaas a whole and a weakness or disappearance of silver lines.This last modification was more intense with Poly I and wasaccompanied by a yellowing of the background - sometimeswith staining of the nuclei of smooth muscle cells. Resultsobtained with proinflammatory albumin were similar whenusing Protocols 1 and 2. Figure 2(d–e) shows some aspectsof the effect of endothelial activation on the pattern of silverstaining.

Neurogenic inflammation

Table 3 shows the modification of the parameters evaluated bysilver staining of the carotid endothelium 4 min after adminis-tration of substance P and 5 min after administration of cap-saicin. In relation to their respective vehicles, both stimuliproduced a discrete decrease in leukocytes which were notfound to adhere preferentially to any of the staining patternspreviously described. Leukocytes were generally in contactwith the interendothelial junction and were sometimes par-tially rimmed by a silver line. Neurogenic inflammation pro-duced in carotid endothelium an increase in argyrophilia asa whole and a weakness or disappearance of silver lines. Infour of six rats substance P produced a marked increase inthe endothelial surface showing a clustering of anionic sites,whereas this phenomenon was discrete in rats injected withthe vehicle. The effect of capsaicin was less notorious. Bothstimuli produced a decrease in transverse lines in relation totheir respective vehicles. Figure 2(f–h) shows some aspects

of the effect of neurogenic inflammation upon the pattern ofsilver staining.

Discussion

In our previous studies in aorta, we never found membranedisruptions in normal rats. Lesive stimuli such as throm-bin and spontaneous pathology produced focalized patchesof wounded endothelial cells in some rats (Gabaldon 1987,Gabaldon et al. 1992), but never long streaks as describedin this study in the carotid. By using albumin as a molecularprobe, Yu & McNeil (1992) identified endothelial cells of therat aorta that incurred and survived transient plasma mem-brane woundsin vivo. These wounded cells were found in allnormal rats as streaks aligned with the long axis of the vessel.Although the authors indicated that internalization of albuminwas a consequence of previous transient plasma membranewounds, these wounds were not exhibited by morphologi-cal methods. In our study silver staining has shown severaltypes of discontinuity of the endothelial pavement. Some ofthese discontinuities formed by short patches of few cellswith intense transverse lines or holes rimmed by gross gran-ularity, may well represent true membrane disruptions. Longstreaks with weak or absent silver lines and rolled endothe-lial nuclei might be formed by intimal foldings secondaryto the vasoconstriction produced by the fixative (Richardsonet al. 1985). The detection of discontinuities in the endothe-lial pavement representing non-artifactual plasma membranedisruptions is an important fact, as endothelial integrity iscentral for preventing platelet deposition and for preservingthe permselective barrier property of the endothelium.

The strategic situation of the endothelium and its activerole in mediating relations between plasma proteins, bloodcells and the subjacent vascular tissue have focused attentionon the importance of a damaged or abnormal endotheliumas a starting point in the pathogenesis of vascular diseasessuch as atherosclerosis. The extraordinary selectivity of silverstaining, which allows the expression of clear-cut differencesbetween neighbors cells, makes it tempting to utilize this tech-nique to recognize an altered or injured endothelium – partic-ularly on taking into account that the literature accumulatedover the years indicates that changes in the silver stainingpattern can be produced by different lesive stimuli (Zaikinaet al. 1982, Dolgovet al. 1982, Klinget al. 1987, Gabaldonet al. 1992, Spanet al. 1992). Given that the endothelium ofcontrol animals expresses different staining patterns, it is dif-ficult to establish a dividing line between what is normal andabnormal, and even more to determine the importance and thedegree of alteration represented by some patterns. We haveadopted an indirect approach to this problem by using stimuliwhich affect endothelial biology in a known manner. A com-mon step in the inflammatory response is the activation of theendothelium; apart from other phenotypic changes, the latterinvolves the expression of receptors implicated in leukocyteadhesion. To activate the endothelium we have chosen twostimuli: a proinflammatory albumin obtained by heat shock,

158 M. Gabaldon & A. Marques

Figure 2. A–C, Plasma membrane disruptions. (A) disappearance of silver lines and intense transverse lines (Protocol 1); (B) absent silver lines, finegranularity and rolled endothelial nuclei (Protocol 2) and (C) holes rimmed by gross granularity (Protocol 2). D–E, Endothelial activation (Protocol1). (D) adhered leukocytes, absence of silver lines and yellowing of the background (albumin) and (E) clump of adhered leukocytes (Poly I). F–H,Neurogenic inflammation (Protocol 2). (F) clustering of anionic sites with disappearance of silver lines (Substance P); (G) gross granularity (SubstanceP) and (H) gross granularity with disappearance of silver lines (capsaicin). Bar, 20µm.

Silver staining of carotid endothelium 159

Table 2. Effect of endothelial activation on theparameters evaluated by silver staining of thecarotid endothelium.

Parameter Albumin Poly I(6) (6)

Stigmata ↓ ↓Fine granularity ↓ ↓Gross granularity ↓ ↓Clustering of anionic sites ↓ ↓Transversal lines n.m. ↓Weak or absent lines ↑ ↑Leukocytes ↑ ↑n.m. = not modified. In parentheses, number ofrats in each group. Silver staining performed withProtocol 1. Results referred to controls.

Table 3. Effect of neurogenic inflammation on theparameters evaluated by silver staining of the carotidendothelium.

Parameter Substance P Capsaicin(6) (7)

Stigmata ↑ ↑Fine granularity ↑ ↓Gross granularity ↑ ↑Clustering of anionic sites ↑ ↑Transversal lines ↓ ↓Weak or absent lines ↑ ↑Leukocytes ↓ ↓In parentheses, number of rats in each group. Silverstaining performed with Protocol 2. Results referred toanimals injected with the vehicle. Vehicle for substanceP was 5 mM acetic acid–0.85% NaCl. Vehicle for cap-saicin was 0.5% ethanol–0.25% Tween 80–0.89% NaCl.

and polyinosinic acid which produces increases in ICAM-1receptors in cultured endothelial cells (Palkamaet al. 1993)and which we have found over years of experience to producethe greatest leukocyte adhesion to the endothelium (Zunigaet al. 1997). Eighteen hours after administration, both stimuliproduced an increase in leukocyte adhesion and a weaknessor disappearance of silver lines.

Endothelial cells contact one another through a number ofsurface molecules organized in different structures (Dejanaet al. 1996). Cell-to-cell adherens junctions are formed bytransmembrane glycoproteins (cadherins) linked to a networkof cytoplasmic/cytoskeletal proteins. These complexes areremarkably dynamic and their composition rapidly changesaccording to the functional state of the cells (Lampugnaniet al. 1992, Dejana 1996). It has been shown that polymor-phonuclear leukocyte adhesion to activated endothelial cellsin culture induces the disorganization of endothelial cell-to-cell adherens junctions and that the same phenomenon is pro-ducedin vivo in postcapillary high endothelial venules withadherent leukocytes (Del Maschioet al. 1996). Althoughthe cellular contacts in the endothelium of the large vesselwe have studied are possibly different to those of culturedor capillary endothelial cells, it is interesting to point out

that leukocyte adhesion to the activated endothelium in ourcase favors the weakening or disappearance of silver lines,and in the other systems the disappearance of the cadherincomplex. Indirect immunofluorescence analysis has shownthat antibodies directed to cadherins yield a fine, continu-ous staining of intercellular margins resembling silver lines(Del Maschioet al. 1996). Cadherins – partially sialylatedglycoproteins – could offer polyanionic residues to uptakecationic silver at sites of endothelial cell contact and mightfunction as nucleation sites for silver grains. Another candi-date for binding silver is the platelet-endothelial cell adhe-sion molecule (PECAM -1), a heavily glycosilated proteinconcentrated at the sites of cell-to-cell contacts that promotehomotypic endothelial cell adhesion in areas distinct fromadherens junctions (Albeldaet al. 1990, Ayalonet al. 1994).As in the case of cadherins, antibodies directed to PECAM-1yield by indirect immunofluorescence a continuous stainingof intercellular junctions resembling silver lines.

Neurogenic inflammation produces in postcapillary venulesan increase in leukocyte adhesion and vascular permeability– the gaps being made visible by silver staining as blackdots (stigmata) along the endothelial cell borders (McDon-ald 1994). We have found none of these effects in carotid,possibly because large vessels are not involved in the inflam-matory response. The intense clustering of anionic sites pro-duced by substance P indicates mobility of the silver-bindingmolecules. Successive steps can be found involving continu-ous silver lines, fragmented silver lines that still delimit theendothelial borders and the complete loss of silver lines withclustering of the anionic sites and the appearance of shortintense hyphen-like figures orientated in the direction of flow.Although neurogenic inflammation produced an increase instigmata, these were not located on the cellular junctionsbut on the endothelial surface. It is well documented thatgross granularity andstigmataon the endothelial junctionsare located in zones of increased permeability (McDonald1994, Baluk & McDonald 1994, Hirataet al. 1995) and arefrequent in mitotic cells (Chuanget al. 1990). The signifi-cance ofstigmataon the endothelial surface is more obscure.It has been suggested that they correspond to myo-endothelialherniae or to multivesicular blebs arising from the endothelialcell membrane (Stetzet al. 1979, Majnoet al. 1985). Man-ifestations of argyrophilia on the endothelial surface havenever been interpreted. Molecules that could bind silver in theendothelial junction may also be present on the endothelialsurface. PECAM-1 is localized over the entire plasma mem-brane (Scholz & Schaper 1997) and some cadherins showan extrajunctional distribution (Salomonet al. 1992) or arestored intracellularly (Lampugnaniet al. 1992).

Silver staining is an old method for examining the mor-phology of endothelial cells. Despite the many studies con-ducted over years (Gottlob & Hoff 1968, Zandet al. 1982,McDonald 1994, Hirataet al. 1995), the cytochemical basis ofsilver nitrate staining remains unknown. It has been proposedthat staining of the interendothelial junctions is a biphasicphenomenon exhibiting certain analogies with the photo-graphic process. Silver cations would thus bind to polyanionic

160 M. Gabaldon & A. Marques

molecules at sites of cell apposition and became reduced tometallic silver which in an autocatalytic process would favorcomplete reduction of the remaining silver. We speculate thatpolyanionic molecules involved in cell-to-cell contacts maybe nucleation sites for the formation of silver lines. The prob-lem is complex for we know neither the nature of silver bind-ing molecules nor the biochemical mechanism involved in theinitial reduction to metallic silver. In this situation, many stud-ies will be required to interpret all the information providedby silver staining, and thus use this technique as a morpho-logical tool for recognizing endothelial alterations.

Acknowledgements

The authors thank Teresa Vicent for her technical assistance.This work was supported by a grant from the Plan Nacional deI + D No. SAF 96–0010. Ana Marques holds a research fel-lowship from the Conselleria de Cultura, Educacio i Ciencia(Generalitat Valenciana, Spain).

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