JPM Vol. 30, No. 3 November 1993:159-162

Prostacyclin Generation by Rat Aortas Obtained by Different Procedures

Maria Gabaldbn, Vicenta Martinez-Sales, Carmen Capdevila, and Angel Ztifiiga

Centro de Znve~tigac~~~, Hospital La Fe, Yalencia, Spain

Keywords: Aorta; Endothelial integrity; Prostacyclin; Silver staining

Endothelium is a monocellular layer very prone to mechanical injury when vessels are handled in vitro. Injury is due to 1) spontaneous vessel shortening and collapse after excision, which produces partial detach- ment of the endothelium from the matrix (Lewis et al., 1991), and 2) obligated manipulations such as the wash- ing off of blood and periadventitial tissue removal. Apart from this type of mechanical damage, the use of solutions inadequate from a physiological point of view may also produce metabolic injury. Morrison et al. (1976, 1977) reported losses of endothelium in rabbit aorta when solutions without oxygen or nut~ents were utilized. In a previous work (Gabaldon and Capdevila, 1991), we studied the effects of different washing solu- tions and of several techniques for removal and clean- ing of the rat aorta on endothelial integrity determined by silver staining of the interendothelial junctions. As prostacyclin is produced by both the endothelium and the medial layer of the aorta (Ts’Ao et al., 1979; Gold- smith, 1982; Boeynaems et al., 1985), loss of endothe- lial integrity in preparation may affect diffusion of the reaction product and alter the overall production of prostacyclin by the vessel.

The aim of the present paper is to establish the effect of aortic processing on endothelial integrity and prosta- cyclin synthesis, utilizing I) different solutions for washing of the aorta; 2) different techniques for re- moval and cleaning of the aorta; and 3) mild in situ

Address reprint requests to Dr. Matia Gabald&, Centro de In- vestigacibn, Avda. Campanar, 21, 46009-Valencia, Spain.

Revised and accepted June 1993.

Journal of Ph~m~coiogical and Toxicological Methods 30, 159-162 (1993) 6 1993 Elsevier Science ~biishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

fixation conditions by perfusion at pressure.

Methods

Removal of the Aorta

The solutions used were as follows

physiological

(mM): 1) Tris 50, NaCl 150, pH 7.4 (Tris-saline); 2) NaCl 118, KC1 4.7, KH*PO, 1.2, MgS04 1.2, NaHC03 25, glucose 5, pH 7.4, gassed with 95% OZ-5% CO2 (Krebs-Ringer); and 3) the same as 2) but with 6% bovine serum albu- min, A-7904, Sigma Chemical Co., (Krebs-Ringer-al- bumin). Ali solutions were utilized at 0°C.

Specific pathogen-free Sprague-Dawley male rats (200-250 g) were used. Animals were anesthetized with 50 mg/kg sodium pentobarbital i.p. Three procedures were utilized for removing the aorta: 1) A 0.8 mm/22- gauge O.D. Vasocan cannula (B. Braum) was inserted in the right common carotid artery, allowing the blood to drain while cutting the abdominal aorta. Once blood pressure decreased markedly, the cannula was fitted to a system perfusing liquid at lo-15 mmHg as described previously (Gabaldon and Capdevila, 1991). Removal of periadventitial tissue and cutting of intercostal arter- ies was performed under a binocular magnifier (2.5 X)

while flushing solutions at low pressure through the aorta. The perfusion solutions were Krebs-Ringer or Krebs-Ringer-albumin; 2) exsanguination, separation of the aorta, and washing off of blood and cleaning of periadventitial tissue in Tris-saline at 0°C by untrained personnel. As a result, occasional stretching and col- lapse occurred; and 3) fixation in situ by perfusion at physiological pressure, as described previously (Ga- baldon, 1987), for 5 min with 1) 2% glutaraldehyde-150

160 JPM Vol. 30, No. 3 November 1993:159-162

mM NaCl-40 mM buffer phosphate, pH 7.4, or 2) the the antibody were as follows: 0.01% to Prostaglandin same fixative with 1% glutaraldehyde at O”C, followed F,, (PGF,,); 0.6% to PGF,,; 0.0025% to PGE,; 0.1% in both cases by a washing off of the fixative with to PGE,; 0.001% to PGD,; and 0.07% to TXBz. The Tris-saline at 0°C for 3 min. Separation of the periad- sensitivity of the assay was 6 pg, and the coefficient ventitial tissue was performed while flushing Tris- of variation was between 9.8% and 12.9% in the range saline at 0°C at low pressure through the aorta. of 5-400 pg. All measurements were made at least in

duplicate. The Student f test was used for statistical analysis of the results.

To avoid manipulations that could damage the endo- thelium, we performed silver staining by immersion of the unopened aortas, hanging them from a hook intro- duced in the orifice of the left common carotid as de- scribed previously (Gabaldon and Capdevila, 1991). Aortas were successively immersed in the following solutions: 1) 2% glutaraldehyde-150 mM NaCl-40 mM phosphate buffer, pH 7.4,lO min; 2) 0.25 M sucrose-20 mM HEPES buffer, pH 7.4 (washing), 1 min; 3) 0.2% AgNOJ-0.1 M sucrose-100 mM HEPES buffer, pH 7.4, 2 min; 4) washing, 1 min; 5) 1% NH4Br, 3 min; 6) washing, 1 min; and 7) 2% glutaraldehyde-6 mM NaCl-0.1 M cacodylate buffer, pH 7.4, 20-24 hr, the first 30 min under a 60-W desk lamp. Aortas were opened between intercostal orifices. Separation of ad- ventitia was performed as described previously (Gabal- don, 1987), and preparations were laid flat and mounted in an aqueous mounting medium. Each speci- men was examined under 400 x magnification by mov- ing the microscopic field systematically along the aorta. The use of a grid eyepiece permitted estimation of the percentage of intact endothelium.

Results

Table 1 shows prostacyclin release by aortas ob- tained by different procedures. Maximum liberation is obtained with procedure B when the rat is exsangui- nated and the aorta is cleaned of periadventitial tissue in Tris-saline by untrained personnel once separated from the animal. Silver staining showed a highly vari- able intact endothelial lining (lo%-60% with a mean of 40%). The remaining surface lacked stained struc- tures or else showed longitudinal and transverse silver lines or a diffuse granularity. Figure 1 shows the silver lines of intact endothelium (a) and the staining pattern of deendothelized areas (b).

Prostacyclin release was notably lowered when aor- tas were handled by procedure A, that is, when vessels were washed off of blood, cleaned of periadventitial tissue while being flushed with Krebs-Ringer with or without albumin and separation was carried out metic- ulously by trained personnel. Differences in prosta- cyclin production with procedures A and B were signif- icant in all cases (p < 0.001).

Stabilization of aortic rings in Krebs-Ringer-albu- min determined the lowest production of prostacyclin, the differences being significant (P < 0.005) with re- spect to Tris-saline. The same effect, though to a lesser extent was observed when stabilization was performed with Krebs-Ringer (p < 0.025). Preser-

Table 1. Prostacyciin Release by Aortas Obtained by Different Procedures

Prostacyclin Release Measurement

The freshly removed thoracic aorta was divided into two parts; the upper portion was utilized for determin- ing endothelial integrity, while the lower part was cut into 3-mm rings. To stabilize enzyme activity and re- duce the influence of manipulation on prostacyclin pro- duction, the rings were kept 45-60 min in Tris-saline, Krebs-Ringer, or Krebs-Ringer-albumin at 4°C. Two rings (5 mg) of aortic tissue were incubated for 15 min at 37°C in 0.5 mL of Tris-saline. The incubation was

pg 6-Kelo-PGF,, No. of

stopped by removing the tissue and adding 10 I.LglmL pg Protein rats

indomethacin. Aliquots of the incubation mixture were Procedure A

stored at - 40°C before assay. The rings were homoge- Krebs-Ringer-albumin” 33 If: 11 5 Krebs-Ringer-albumi@ 621 5

nized in 1.15% KC1 (1: 200 w/v); 2% deoxycholate was Krebs-Ringer” 17 t: 3 5

added to the aortic homogenate to solubilize particu- Procedure B” 109 t: 18 6

late protein prior for its determination. Total protein procedure C”

content was, in turn, determined by the method de- Glutaraldehyde 2%, 22°C 11 2 4 S

scribed by Lowry et al. (1951). Glutaraldehyde I%, 0°C 34 rt 8 5

6 Keto-PGFi, (the stable hydrolysis product of n Stabilization in Tris-saline.

prostacyclin) was measured by radioimmunoassay b Stabilization in Krebs-Ringer-albumin.

(RIA) according to Maclouf (1982) using specific anti- c Stabilization in Krebs-Ringer. Note: Results are expressed as mean rt S.D. See Methods for

bodies obtained in our laboratory. Cross-reactivities of description of the procedures.

M. GABALDiIN ET AL. f6f AORTA HANDLING AND PROSTACYCLIN

Figure 1. Different patterns of silver staining. 1) Intact endo- thelium in aortas obtained by procedure A and flushed with Krebs-Ringer-albumin; 2) deendothelized area in aorta re- moved by procedure B. Scale bar = 20 km.

vation of endothelial integrity was maximum with Krebs-Ringer albumin, where 95% of the endothelium was intact. Preservation was a little less with Krebs-Ringer (8.5%), due to the lack of protection ex- erted by the ~bumin regarding mechanical injury caused by manipulation or collapse (Gabafd~n and Capdevila, 1991).

Prostacyclin release was similar in aortas fixed with 1% giutaraldehyde at 0°C for 5 min and in aortas ob- tained with Krebs-Ringer-albumin when Tris-saline was utilized during stabilization. Stronger fixation by 2% glutaraldehyde at 22°C caused a decrease in prosta- cyclin release similar to that obtained by procedure A when stabilization was performed in Krebs-Ringer.

Discussion Handling of bfood vessels with preservation of en-

dothelial integrity is imperative in 1) preparation of ves-

sels for grafting, as partial deendothelization increases the risk of early occlusion by thrombi or late occlusion by fibrocellular intimal thickening; 2) pharmacological studies with agonists that act on smooth muscle through mediators released by the endothelium; and 3) biochemical studies on whole vessels when enzyme activities are expressed in the intimal and medial lay- ers. To preserve endothelial integrity we used princi- ples of endothelial cell culture to secure optimal condi- tions for the survival of the endothelium, such as glucose-containing, oxygenated, and balanced salt solutions supplemented with albumin. These media have been shown by others to be the most adequate to prevent denudation during handling of vessels (Mor- rison et al., 1976, 1977; Haudenschild et al., 1981).

Conflicting reports have been published concerning the release of prostacyclin by deendothelized vessels. Although, in general, deendotheiization produces a greater prostacyclin liberation (Goldsmith, 1982; Boey- naems et al., 1985; Mehta et al., 1982), other cases have been described in which prostacychn either is not affected (Ts’Ao et al., 1979) or does diminish (Grabow- ski et al., 1985; Rosolowsky et al., 1991; Elder et al., 1981). Some methods for removing aortic endothelium may damage smooth muscle cells, and this affects ara- chidonic acid metabolism (Rosolowsky et al., 1991).

In our study, deendothelization is not complete, but partial-this being a consequence of inadequate han- dling of the vessel. In studies with deendothehzed ves- sels, prostacychn is produced exclusively by the me- dial layer. In our study, three sources of prostacyclin coexist: the medial layer in zones of frank denudation, the normal endothelium, and the damaged endothehum exhibiting a high degree of disorganization in which sloughed endothelial cells are still partially attached to the basal membrane (Gabaldon and Capdevila, 1991). We do not know whether this disorganized endothe- lium is hyper- or hypofunctional with respect to prosta- cyclin generation. It has been shown that in endothelial cell cultures different incubation media produce prost- acyclin release which parallels the release of 5’Cr (Brox and Nordoy, 1982). Because the latter is regarded as an indicator of celi membrane damage, these results seem to indicate that cell injury increases prostacychn production.

In our study mechanical damage, anoxia, and lack of nutrients were found to produce a variable loss of endothelium and a burst in prostacyclin production. As mechanical and biochemical injuries to the endothe- lium may compromise results in determining prosta- cyclin generation, we recommend I) the use of solu- tions that preserve endothehal integrity for both re- moval of the vessel and the stabilization period; and 2) the assessment of correct manipulation by AgN03 staining or SEM.

162 JPM Vol. 30, No. 3 November 1993:159-162

This study was supported by a grant no. 87/1030 from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS), Spain. The authors gratefully acknowledge the help of Maria Luisa Aragones, Guadalupe Manzano, and Teresa Vicent for their technical assis- tance.

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