Histochemistry 53, 217-222 (1977)
Histochemistry 9 by Springer-Verlag 1977
Vascular Reactions to Horseradish Peroxidase in the Guinea Pig Torgeir Vegge and Rolf Haye Anatomical Institute, University of Oslo, and Ear, Nose, Throat Department, Rikshospitalet, Karl Johansgt. 47,Oslo, Norway
Summary. Albino guinea pigs were given intradermal injections of the protein tracer horseradish peroxidase. In a 0.l mM concentration the tracer did not increase vascular permeability to Evans blue-labelled plasma proteins. In a 1 mM concentration, however, the peroxidase induced a local vascular leakage. This leakage was almost totally inhibited by pretreating the animals with acetylsalicylic acid, while antihistamine had only a weak inhibitory effect. We therefore believe that prostaglandins are important mediators in this HRP-induced vascular reaction.
Introduction Since horseradish peroxidase (HRP) was introduced as a protein tracer for electron microscopy a decade ago (Karnovsky and Graham, 1966), it has gained wide use in the investigation of epithelial and vascular permeability. Its popularity stems from the combination of its relatively small molecular size with easy visualization. It was soon recognized that in certain species this tracer caused increased vascular permeability, thus making the interpretation of observations difficult (Karnovsky and Cotran, 1966). Cotran and Karnovsky (1967) investigated this effect, and found that HRP caused increased vascular permeability in rats. This effect could be inhibited by treating the animal with a combination of antihistamine and antiserotonin. They also found increased permeability in guinea pig vessels, but did not investigate this in detail. They found no increased permeability in mice, but were later contradicted on this point by Clementi (1970) and by Williams and Wissig (1975). Furthermore, Winther (1971) did not detect increased blood levels of histamine in guinea pigs after intravenous H R P injections. In several experiments we have used H R P as a tracer in albino guinea pigs without observing untoward effects (Haye, 1972; Haye and Vegge, 1973). These inconsistencies made it desirable to reinvestigate the permeabilityincreasing effect of H R P on guinea pig vessels.
218
T. Vegge and R. Haye
Material and Methods Albino guinea pigs of a local strain (SFF: PON) of either sex weighing 350450 g were used. The abdominal skin was clipped, and the animals were anesthetized with 1 mg Nembutal intraperitoneally. Evans Blue (0.5 ml of a 0.5% solution) was injected into a jugular vein. After 10 rain, skin tests were performed by injecting test substances in 30 gl volumes intradermally on the abdomen. Injections were placed in antero-posterior rows about 20 mm to either side of the midline, up to 3 injections in each row, The animlas were killed with Nembutal intracardially 20 min after the intradermal injections, and the leakage of Evans Blue at the injection sites was assessed by measuring the diameter and estimating the intensity of blue staining on the inside of the skin. Eight animals were pretreated with 80 mg acetylsalicylic acid intraperitoneally 30 min prior to the skin tests. The acetylsalicylic acid was dissolved with 72 mg L-lysin in 2 ml sterile water. 8 control animals were pretreated with 72 mg L-lysine.
Substances Used. Horseradish peroxidase, types II and VI (Sigma Chemical Co.); human serum albumin (Kabi, Stockholm, Sweden); 5-hydroxytryptamine creatinine sulfate complex (serotonin, Sigma Chemical Co.); Histamine dihydrochloride; pyrilamine maleate (NF) ; methysergide (Sandoz, Basel). Methysergide was supplied free of charge by the manufacturers. Their generosity is gratefully acknowledged.
Results
The volume of the skin injections caused a local swelling of about 5 mm diameter. When the injected substance caused an increased vascular permeability, a blue stain appeared at the site of injection with a minimum diameter corresponding to this injected swelling. The intensity of the color varied. Therefore, we rated the reactions on a scale from 0 to 5-t-, taking both intensity and size into consideration. The strongest reaction, both in diameter and in intensity of color, was caused by 1 gg of histamine (Fig. 1). This gave an intensely blue stain of 10-15ram diameter which was given a rating of 5 + . A stain of almost the same diameter but with less intensity was given a 4 + or 3 + rating, while the residual pin-prick spot caused by e.g. the saline controls, was rated 0 (Fig. 1). Neither H R P II nor H R P VI (horseradish peroxidase, type II or VI) caused any discernible permeability increase when injected in a 0.1 mM concentration.
Figs. 1-4. Abdominal skin showing Evans Blue staining after skin tests. The skins have been removed and mounted for photography. In each figure are three injections: 1 p.g histamine left, saline control center, and 1.2 mg (1 mM conc.) HRP II right. Arrows point to injection sites. Magnification xl.3 Fig. 1. Untreated animal. HRP-induced stain (right) almost identical to histamine-induced stain Fig. 2. Untreated animal, 10 gg pyrilamine maleate added to each injection. Note complete blocking of histamine stain, very slight effect on HRP stain Fig. 3. Animal pretreated with acetylsalicylic acid. Complete inhibition of HRP effect, slight reduction of histamine effect Fig. 4. Animal pretreated with aeetylsalicylic acid, 10 gg pyrilamine maleate added to each injection. Complete inhibition of both HRP- and histamine-induced leakage
~v
©
O
220
T. Vegge and R. Haye
Table 1. Effect of various substances on vascular permeability as determined by skin tests (number of animals in brackets) Substance
Permeability rating
Substance
Permeability rating
Histamine, 1 l.tg H R P II, 0.1 m M H R P VI, 0.1 m M H R P II, 1 m M H R P VI, 1 m M HSA, 0.1 m M
5 + (55) 0 (8) 0 (7) 4 + (55) 3 + (7) 0 (11)
HSA, 1 m M 5-HTA, 0.i lag 5-HTA, 1 btg 5-HTA, 10 lag saline
0 0 0 0 0
(7) (3) (3) (3) (55)
Table 2. Inhibitory effect of antihistamine, antiserotonin, and acetylsalicylic acid on the HRP- and histamine-induced vascular leakage as determined by skin tests (number of animals in brackets) Substance
Permeability rating No pretreatment
H R P II 1 m M H R P II 1 mM, pyrilamine maleate 10 btg H R P II 1 mM, methysergide l i.tg H R P II 1 mM, pyrilamine maleate 10 lag, methysergide 1 lag Histamine 1 lag, pyrilamine maleate 10 lag Pyrilamine maleate 10 lag Methysergide 1 ; g Saline
Pretreated with 80 mg acetylsalicylic acid
4 + (55)
1+ (s)
3+ (11) 4+ (7) 3+ (7)
0
(8)
0 o
(18) (18)
0 o
(a) (8)
o 0
(7) (55)
0
(8)
The same was true for HSA (human serum albumin), and for 5-HTA (5-hydroxytryptamine). However, in a 1.0 mM concentration, H R P caused considerable increase in vascular permeability, somewhat greater with H R P II than with H R P VI (Table 1). In an attempt to clarify the mechanisms responsible for the HRP-induced vascular leakage antihistamine (pyrilamine maleate) and antiserotonin (methysergide), either alone or in combination were added to the HRP-II injections. The results are listed in Table 2, and it will be seen that antihistamine had only a slight inhibitory effect on the HRP-induced leakage, while antiserotonin had no effect. The same dose of antihistamine completely inhibited the vascular leakage caused by 1 lag of histamine (Fig. 2). In animals pretreated with acetylsalicylic acid the HRP-induced permeability increase was reduced to a minimum (Figs. 3 and 4). The recidual leakage was eliminated by pyrilamine maleate (Table 2). Injections of lysine in 8 control animals had no effect.
Vascular Reactions to Peroxidase
221
Discussion
Cotran and Karnovsky (1967) demonstrated that after injection of HRP II into the skin of Sprague-Dawley rats, an effusion of previously injected marker dye occurred, indicating increased local vascular leakage of plasma proteins. These authors also reported limited experiments on guinea pigs with similar results. In the present study, we detected no increased vascular leakage after local application of horseradish peroxidase, when used in a 0.1 mM concentration. This concentration is similar to the intravascular concentrations of HRP II often used in permeability studies (Haye and Vegge, 1973; Sugar etal., 1972; Karnovsky, 1967; Winther, 1971 ; William and Wissig, 1975). It should be kept in mind that these experiments involve the presentation of the test substance to the outside of the vessels, while in permeability studies it is usually presented to the inside. However, the poor reputation of HRP II as a tracer in guinea pigs is based on the same type of tests (Cotran and Karnovsky, 1967). Judged by these criteria and under these circumstances, H R P does not cause permeability increase in the guinea pig. This is supported by experiments with H R P II given intracardially to unanesthetised animals, where we found no adverse effects (Haye et al., in prep.), contrasting the observations made in rats by Cotran and Karnovsky (1967). In higher concentration (1 mM) H R P II caused a definite vascular leakage. When H R P VI was used, the leakage was still present, but less conspicuous. Human serum albumin caused no leakage. The leakage, therefore, is not brought about by the protein concentration per se. In rats the HRP-induced vascular leakage was inhibited by a combination of antihistamine and antiserotonin (Cotran and Karnovsky, 1967). In the present study we have demonstrated that, in guinea pigs, the HRP-induced leakage is not significantly reduced by antihistamine or antiserotonin, either alone or combined. The effect of acetylsalicylic acid on prostaglandin synthesis is well known (Vane, 1971). By pretreating the animals with acetylsalicylic acid we obtained almost complete inhibition of the vascular leakage. In our opinion, this is strong evidence, if not conclusive proof, that prostaglandins mediate the vascular permeability increase induced by high doses of HRP in the guinea pig. Our experiments demonstrate that the effect of H R P on capillary permeability in the guinea pig is dose-related. In our strain a moderate dose of HRP II did not cause any leakage. With proper controls and in appropriate dosage, this substance may therefore be used as a vascular tracer in guinea pigs. References Clementi, F. : Effect of horseradish peroxidase on mice lung capillaries' permeability. J. Histochem. Cytochem. 18, 887 892 (1970) Cotran, R.S., Karnovsky, M.J.: Vascular leakage induced by horseradish peroxidase in the rat. Proc. Soc. exp. Biol. (N.Y.) 126, 557 561 (1967)
222
T. Vegge and R. Haye
Haye, R., Vegge, T. : The capillaries of the middle ear mucosa in the guinea pig. Z. Zellforsch. 143, 517-526 (1973) Haye, R., Vegge, T., Sjaastad, O.: Effect of horseradish peroxidase on the blood pressure of guinea pigs and rabbits (in prep.) Karnovsky, M.J.: The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35, 213-236 (1967) Karnovsky, M.J., Cotran, R.S. : The intercellular passage of exogenous peroxidase across endothelium and mesothelium. Anat. Rec. 154, 365 (1966) Karnovsky, M.J., Graham, R.C. : A new cytochemical method for peroxidase as a protein tracer in electron microscopy. International Symposium on Electron Microscopy and Cytochemistry, Leiden, 1966 Sugar, J.O., Engstr6m, H., Stable, J.: Stria vascularis. Acta Otolaryng. Suppl. 301, 61-73 (1972) Vane, J.R. : Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature [New Biol.] 231,232 (1971) Williams, M.C.,Wissig, S.L.: The permeability of muscle capillaries to horseradish peroxidase. J. Cell Biol. 66, 531 555 (1975) Winther, F.O.: The permeability of the guinea pig cochlear capillaries to horseradish peroxidase. Z. Zellforsch. 114, 193-202 (1971)
Received May 12, 1977
Histochemistry 9 by Springer-Verlag 1977
Vascular Reactions to Horseradish Peroxidase in the Guinea Pig Torgeir Vegge and Rolf Haye Anatomical Institute, University of Oslo, and Ear, Nose, Throat Department, Rikshospitalet, Karl Johansgt. 47,Oslo, Norway
Summary. Albino guinea pigs were given intradermal injections of the protein tracer horseradish peroxidase. In a 0.l mM concentration the tracer did not increase vascular permeability to Evans blue-labelled plasma proteins. In a 1 mM concentration, however, the peroxidase induced a local vascular leakage. This leakage was almost totally inhibited by pretreating the animals with acetylsalicylic acid, while antihistamine had only a weak inhibitory effect. We therefore believe that prostaglandins are important mediators in this HRP-induced vascular reaction.
Introduction Since horseradish peroxidase (HRP) was introduced as a protein tracer for electron microscopy a decade ago (Karnovsky and Graham, 1966), it has gained wide use in the investigation of epithelial and vascular permeability. Its popularity stems from the combination of its relatively small molecular size with easy visualization. It was soon recognized that in certain species this tracer caused increased vascular permeability, thus making the interpretation of observations difficult (Karnovsky and Cotran, 1966). Cotran and Karnovsky (1967) investigated this effect, and found that HRP caused increased vascular permeability in rats. This effect could be inhibited by treating the animal with a combination of antihistamine and antiserotonin. They also found increased permeability in guinea pig vessels, but did not investigate this in detail. They found no increased permeability in mice, but were later contradicted on this point by Clementi (1970) and by Williams and Wissig (1975). Furthermore, Winther (1971) did not detect increased blood levels of histamine in guinea pigs after intravenous H R P injections. In several experiments we have used H R P as a tracer in albino guinea pigs without observing untoward effects (Haye, 1972; Haye and Vegge, 1973). These inconsistencies made it desirable to reinvestigate the permeabilityincreasing effect of H R P on guinea pig vessels.
218
T. Vegge and R. Haye
Material and Methods Albino guinea pigs of a local strain (SFF: PON) of either sex weighing 350450 g were used. The abdominal skin was clipped, and the animals were anesthetized with 1 mg Nembutal intraperitoneally. Evans Blue (0.5 ml of a 0.5% solution) was injected into a jugular vein. After 10 rain, skin tests were performed by injecting test substances in 30 gl volumes intradermally on the abdomen. Injections were placed in antero-posterior rows about 20 mm to either side of the midline, up to 3 injections in each row, The animlas were killed with Nembutal intracardially 20 min after the intradermal injections, and the leakage of Evans Blue at the injection sites was assessed by measuring the diameter and estimating the intensity of blue staining on the inside of the skin. Eight animals were pretreated with 80 mg acetylsalicylic acid intraperitoneally 30 min prior to the skin tests. The acetylsalicylic acid was dissolved with 72 mg L-lysin in 2 ml sterile water. 8 control animals were pretreated with 72 mg L-lysine.
Substances Used. Horseradish peroxidase, types II and VI (Sigma Chemical Co.); human serum albumin (Kabi, Stockholm, Sweden); 5-hydroxytryptamine creatinine sulfate complex (serotonin, Sigma Chemical Co.); Histamine dihydrochloride; pyrilamine maleate (NF) ; methysergide (Sandoz, Basel). Methysergide was supplied free of charge by the manufacturers. Their generosity is gratefully acknowledged.
Results
The volume of the skin injections caused a local swelling of about 5 mm diameter. When the injected substance caused an increased vascular permeability, a blue stain appeared at the site of injection with a minimum diameter corresponding to this injected swelling. The intensity of the color varied. Therefore, we rated the reactions on a scale from 0 to 5-t-, taking both intensity and size into consideration. The strongest reaction, both in diameter and in intensity of color, was caused by 1 gg of histamine (Fig. 1). This gave an intensely blue stain of 10-15ram diameter which was given a rating of 5 + . A stain of almost the same diameter but with less intensity was given a 4 + or 3 + rating, while the residual pin-prick spot caused by e.g. the saline controls, was rated 0 (Fig. 1). Neither H R P II nor H R P VI (horseradish peroxidase, type II or VI) caused any discernible permeability increase when injected in a 0.1 mM concentration.
Figs. 1-4. Abdominal skin showing Evans Blue staining after skin tests. The skins have been removed and mounted for photography. In each figure are three injections: 1 p.g histamine left, saline control center, and 1.2 mg (1 mM conc.) HRP II right. Arrows point to injection sites. Magnification xl.3 Fig. 1. Untreated animal. HRP-induced stain (right) almost identical to histamine-induced stain Fig. 2. Untreated animal, 10 gg pyrilamine maleate added to each injection. Note complete blocking of histamine stain, very slight effect on HRP stain Fig. 3. Animal pretreated with acetylsalicylic acid. Complete inhibition of HRP effect, slight reduction of histamine effect Fig. 4. Animal pretreated with aeetylsalicylic acid, 10 gg pyrilamine maleate added to each injection. Complete inhibition of both HRP- and histamine-induced leakage
~v
©
O
220
T. Vegge and R. Haye
Table 1. Effect of various substances on vascular permeability as determined by skin tests (number of animals in brackets) Substance
Permeability rating
Substance
Permeability rating
Histamine, 1 l.tg H R P II, 0.1 m M H R P VI, 0.1 m M H R P II, 1 m M H R P VI, 1 m M HSA, 0.1 m M
5 + (55) 0 (8) 0 (7) 4 + (55) 3 + (7) 0 (11)
HSA, 1 m M 5-HTA, 0.i lag 5-HTA, 1 btg 5-HTA, 10 lag saline
0 0 0 0 0
(7) (3) (3) (3) (55)
Table 2. Inhibitory effect of antihistamine, antiserotonin, and acetylsalicylic acid on the HRP- and histamine-induced vascular leakage as determined by skin tests (number of animals in brackets) Substance
Permeability rating No pretreatment
H R P II 1 m M H R P II 1 mM, pyrilamine maleate 10 btg H R P II 1 mM, methysergide l i.tg H R P II 1 mM, pyrilamine maleate 10 lag, methysergide 1 lag Histamine 1 lag, pyrilamine maleate 10 lag Pyrilamine maleate 10 lag Methysergide 1 ; g Saline
Pretreated with 80 mg acetylsalicylic acid
4 + (55)
1+ (s)
3+ (11) 4+ (7) 3+ (7)
0
(8)
0 o
(18) (18)
0 o
(a) (8)
o 0
(7) (55)
0
(8)
The same was true for HSA (human serum albumin), and for 5-HTA (5-hydroxytryptamine). However, in a 1.0 mM concentration, H R P caused considerable increase in vascular permeability, somewhat greater with H R P II than with H R P VI (Table 1). In an attempt to clarify the mechanisms responsible for the HRP-induced vascular leakage antihistamine (pyrilamine maleate) and antiserotonin (methysergide), either alone or in combination were added to the HRP-II injections. The results are listed in Table 2, and it will be seen that antihistamine had only a slight inhibitory effect on the HRP-induced leakage, while antiserotonin had no effect. The same dose of antihistamine completely inhibited the vascular leakage caused by 1 lag of histamine (Fig. 2). In animals pretreated with acetylsalicylic acid the HRP-induced permeability increase was reduced to a minimum (Figs. 3 and 4). The recidual leakage was eliminated by pyrilamine maleate (Table 2). Injections of lysine in 8 control animals had no effect.
Vascular Reactions to Peroxidase
221
Discussion
Cotran and Karnovsky (1967) demonstrated that after injection of HRP II into the skin of Sprague-Dawley rats, an effusion of previously injected marker dye occurred, indicating increased local vascular leakage of plasma proteins. These authors also reported limited experiments on guinea pigs with similar results. In the present study, we detected no increased vascular leakage after local application of horseradish peroxidase, when used in a 0.1 mM concentration. This concentration is similar to the intravascular concentrations of HRP II often used in permeability studies (Haye and Vegge, 1973; Sugar etal., 1972; Karnovsky, 1967; Winther, 1971 ; William and Wissig, 1975). It should be kept in mind that these experiments involve the presentation of the test substance to the outside of the vessels, while in permeability studies it is usually presented to the inside. However, the poor reputation of HRP II as a tracer in guinea pigs is based on the same type of tests (Cotran and Karnovsky, 1967). Judged by these criteria and under these circumstances, H R P does not cause permeability increase in the guinea pig. This is supported by experiments with H R P II given intracardially to unanesthetised animals, where we found no adverse effects (Haye et al., in prep.), contrasting the observations made in rats by Cotran and Karnovsky (1967). In higher concentration (1 mM) H R P II caused a definite vascular leakage. When H R P VI was used, the leakage was still present, but less conspicuous. Human serum albumin caused no leakage. The leakage, therefore, is not brought about by the protein concentration per se. In rats the HRP-induced vascular leakage was inhibited by a combination of antihistamine and antiserotonin (Cotran and Karnovsky, 1967). In the present study we have demonstrated that, in guinea pigs, the HRP-induced leakage is not significantly reduced by antihistamine or antiserotonin, either alone or combined. The effect of acetylsalicylic acid on prostaglandin synthesis is well known (Vane, 1971). By pretreating the animals with acetylsalicylic acid we obtained almost complete inhibition of the vascular leakage. In our opinion, this is strong evidence, if not conclusive proof, that prostaglandins mediate the vascular permeability increase induced by high doses of HRP in the guinea pig. Our experiments demonstrate that the effect of H R P on capillary permeability in the guinea pig is dose-related. In our strain a moderate dose of HRP II did not cause any leakage. With proper controls and in appropriate dosage, this substance may therefore be used as a vascular tracer in guinea pigs. References Clementi, F. : Effect of horseradish peroxidase on mice lung capillaries' permeability. J. Histochem. Cytochem. 18, 887 892 (1970) Cotran, R.S., Karnovsky, M.J.: Vascular leakage induced by horseradish peroxidase in the rat. Proc. Soc. exp. Biol. (N.Y.) 126, 557 561 (1967)
222
T. Vegge and R. Haye
Haye, R., Vegge, T. : The capillaries of the middle ear mucosa in the guinea pig. Z. Zellforsch. 143, 517-526 (1973) Haye, R., Vegge, T., Sjaastad, O.: Effect of horseradish peroxidase on the blood pressure of guinea pigs and rabbits (in prep.) Karnovsky, M.J.: The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35, 213-236 (1967) Karnovsky, M.J., Cotran, R.S. : The intercellular passage of exogenous peroxidase across endothelium and mesothelium. Anat. Rec. 154, 365 (1966) Karnovsky, M.J., Graham, R.C. : A new cytochemical method for peroxidase as a protein tracer in electron microscopy. International Symposium on Electron Microscopy and Cytochemistry, Leiden, 1966 Sugar, J.O., Engstr6m, H., Stable, J.: Stria vascularis. Acta Otolaryng. Suppl. 301, 61-73 (1972) Vane, J.R. : Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature [New Biol.] 231,232 (1971) Williams, M.C.,Wissig, S.L.: The permeability of muscle capillaries to horseradish peroxidase. J. Cell Biol. 66, 531 555 (1975) Winther, F.O.: The permeability of the guinea pig cochlear capillaries to horseradish peroxidase. Z. Zellforsch. 114, 193-202 (1971)
Received May 12, 1977
