rabbits were paired and both strains were used for For the estimation of mediators in the grafts, rabbits

Transcription

1 Br. J. Pharma. (1978), 64, HANGES N BLOOD FLOW AND MEDATOR ONTENT OF RABBT SKN GRAFTS G.P. LEWS & BEVERLEY A. MANGHAM Department of Pharmaology, Royal ollege of Surgeons, Linoln's nn Fields, London W2A 3PN 1 Blood flow hanges have been measured in rabbit skin homografts and autografts and an attempt has been made to orrelate these hanges with the presene of vasoative agents in homogenates of the grafts. 2 During the healing-in of both types of grafts the blood flow and histamine ontent inreased. 3 f this histamine was responsible for the vasodilatation via an H2-reeptor it must have been released in the form of 'nasent' or intrinsi histamine sine the vasodilatation was not antagonized by antihistamine. 4 A peak of histamine onentration ourred in homografts, more than three times that in autografts at the onset of rejetion. At the same time the blood flow stopped ompletely. 5 This vasoonstrition might be mediated by histamine sine treatment with mepyramine delayed the essation of blood flow from 5 days up to 7 to 1 days. 6 Prostaglandins appeared to be involved only in the period of peak blood flow in homografts beause indomethain did not delay the onset of rejetion, but redued the peak blood flow in the homografts at a time when there was an inreased ontent of prostaglandin E2 in the homograft tissue. ntrodution When skin is transplanted as either autografts or homografts, distint vasular hanges our during healing-in and in the ase of the homograft also at the onset of rejetion (Medawar, 1944). The question arises, do these vasular hanges reflet the ativity of pharmaologial mediators? To investigate this possibility, an attempt was made to orrelate the blood flow hanges whih our in grafted skin with the presene of mediators in homogenates of the graft tissue. Methods Skin graft proedure Adult New Zealand (NZ) and Norfolk White (NF) rabbits weighing 3. to 4. kg anaesthetized with sodium pentobarbitone (4 mg/kg i.v.) were used. Full thikness skin grafts (2. x 2. m) were removed from the hind limbs and either transplanted onto the hind limbs or onto the bak from whih similar size pathes of dorsal skin had been removed (Jasani & Lewis, 1971). For the homografts the NZ and NF rabbits were paired and both strains were used for the autografts. olletion, homogenization and extration of tissues For the estimation of mediators in the grafts, rabbits reeived either 12 homografts of leg skin transplanted onto the bak, or 5 autografts of leg skin transplanted onto the legs. At various times after grafting, the sutures were arefully removed and the graft was torn from the graft bed with a sturdy pair of toothed foreps and instantly frozen in alohol and dry ie. Pressure was maintained on the graft bed with a swab until bleeding had stopped (usually 2 to 3 min). The removed grafts were blotted dry, weighed and wrapped in tin foil and stored at -2 until they were extrated. Non-operated skin was removed to provide ontrols and treated in the same manner as the grafts. For homogenization the tissues were thawed, finely hopped with sissors and suspended in 2 ml of ie-old 6% ethanol, and homogenized at 4 with a Polytron Ultra Turax homogenizer run at 3% of the maximum speed for 9 seonds. The homogenizer was washed and the washings added to

2 124 G.P. LEWS & BEVERLEY A. MANGHAM the homogenate, with a further 2 ml of ie-old 6% ethanol. The homogenate was entrifuged at 5, rev/min for 3 min to obtain a lear supernatant. The supernatant was poured into a 25 ml evaporating flask and the pellet was resuspended in 3 ml of 6% ethanol and mixed on a vortex rotor for 6 s and entrifuged. The supernatants were pooled and the pellet was disarded. The extrat was evaporated to dryness under a partial vauum at 37 in a Rotavapor-R (Buhi). The dried extrat was dissolved in 4ml of saline (.9% w/v Nal solution) and divided into 1 ml aliquots in plasti tubes, apped and stored at -2 until they were assayed. These samples were assayed diret, with no further purifiation exept for the prostaglandin assay. Some samples were further extrated for prostaglandins (Gilmore, Vane & Wyllie, 1968) to hek that the protein in the samples did not affet the results of the radioimmunoassay. Assay of mediators Eah tissue extrat was assayed for prostaglandins E2 and F2,,, histamine and 5-hydroxytryptamine (5-HT). A radioimmunoassay was used for the prostaglandins (Hennam, Johnson, Newton & ollins, 1974). Before the histamine and 5-HT ontents were assayed, the protein in eah sample was preipitated with perhlori aid and the supernatant was assayed by an automated fluorimetri tehnique (Bogdanski, Pletsher, Brodie & Udenfriend, 1956; Shore, Burkhalter & ohen, 1959; Maikel & Miller, 1966; Evans, Lewis & Thomson, 1973). Blood flow measurement For all blood flow measurements, the skin grafts were transplanted from the legs onto the bak, eah animal reeived 12 grafts and both autografts and homografts were studied. Eah rabbit was restrained in a box, and a miro injetion of "'Xenon in saline (.1 ml) was given intradermally to normal skin or grafted tissue. The washout of radioativity was monitored for at least 3 min with a ollimated y-sintillation detetor (Lewis, Pek, Williams & Young, 1976). The detetor, a sodium iodide thalium ativated rystal was plaed diretly above the site of injetion, about 1 m from the surfae of the rabbit. The washout was reorded every 4 s and printed automatially on a Panax reorder. The '33Xe was diluted aording to its age to give an initial ount of at least 35, but not greater than 8, ounts in the first 4 s period. The results were expressed as a perentage of the initial ount and plotted on a semi-logarithmi sale against time. The urve peeling tehnique was used to draw two exponentials from the urve, and the b o D o 3 m ~ Wl Figure 1 Blood flow hanges in rabbit skin autografts (A) and homografts (). The blood flow is expressed in terms of the learane onstant (K) for Xenon, K =.6932 Eah point represents the mean of 4 experiments for autografts, 7 experiments for homografts; vertial lines show s.e. means. No blood flow ould be deteted before day 3, at whih time it was similar to that of normal skin (o A). n the autograft, the blood flow inreased forming a plateau between day 4 and 6, and subsequently dereased to normal skin blood flow. n the homograft the plateau of high blood flow was maintained for 12 h only, after whih no further blood flow ould be deteted. half times (T1/2) for '33Xe in the skin and in the subutaneous tissue were alulated (Sejrsen, 1969). From these half times the learane onstant, K was alulated giving an indiret measurement of blood flow in the tissue. All blood flows will be expressed in terms of K. The effet of several pharmaologial antagonists on the blood flow of both the homograft and the autograft was measured by the '3"Xenon learane method. The following antagonists were given intravenously in divided doses from the first or third day after grafting: indomethain (2 x 5 and 1 mg kg-' day-1), methysergide (3 x.3 mg kg-' day-'), mepyramine (3 x 2.5 mg kg-' day-'), metiamide (3 x 5 mg kg'- day- 1), and metiamide plus mepyramine (3 x mg kg-' day-'). Results Blood flow hanges The blood flow measurements were made in animals in whih 12 leg skin autografts or homografts had been transplanted onto the bak. The blood flow

3 BLOOD FLOW AND MEDATORS N GRAFTS 125 U1) ) a, (5 U1) *1 lo O Figure 2 Mediator ontent of rabbit skin autografts. A total of 5 grafts were transplanted onto eah animal. Eah point represents the mean of at least 8 samples; vertial lines show s.e. means. There was no signifiant hange in the tissue ontent of prostaglandin E2 (PGE2, ), prostaglandin F2a (PGF29 A), and 5-hydroxytryptamine (5-HT, A) all expressed as ng/g tissue. However, the histamine ontent as gg/g tissue (o) inreased signifiantly at day 3-4 and formed a plateau between day 5 and 7, after whih the histamine ontent dereased to that of normal skin. The ontent of ontrol skin was histamine gg/g, 5-HT ng/g, PGE ng/g, PGF ng/g. -a3. U1) 3 ) D ) O 15 '- () 1 5 't s 't E 'a O S D (a ) ioo 'O o ) U) o Figure 3 Mediator ontent of rabbit skin homografts. All animals eah reeived a total of 12 grafts. Eah point represents the mean of 4 samples; vertial lines show s.e. means. The histamine ontent as pg/g tissue () formed 2 peaks, the first oinided with a signifiant short-lived plateau of prostaglandin E2 (PG E2) as ng/g tissue (). The seond ourred together with inreases in PGE2 and prostaglandin F2Q (PGF2a, A) after day 5 i.e. after rejetion of the homograft. There was no signifiant hange in the 5-hydroxytryptamine (5-HT) ontent (A). The ontent of ontrol skin was histamine 2.1T +.23 glg/g, 5-HT ng/g, PGE ng/g, PGF ng/g. L expressed in terms of the learane onstant for 133Xenon is plotted against days after grafting in Figure 1. No blood flow was deteted in either the autografts or the homografts until day 3 at whih time it was similar to that of normal skin. Between days 3 and 4 it inreased rapidly reahing a peak around day 4 in both autografts and homografts. n autografts the peak flow was maintained until day 6, after whih the flow gradually returned to normal during the following 4 to 6 days. n the homografts, the peak flow was maintained for only a 12 h period. This was followed by a sudden fall in flow and at 5 days, 6 h the flow had eased altogether. Mediator ontent of autografts At daily intervals up to 9 days after grafting all grafts were removed from one animal at the same time and proessed separately. The ontent of the different mediators in the autografts is illustrated in Figure 2. During the healing-in of the autograft over the first 4 days after grafting, the ontents of prostaglandin E2 (PGE2), PGF2a and 5-HT levelled off to values found in normal skin and remained within those levels up to day 9. The histamine ontent, however, inreased signifiantly on day 4 and ontinued to inrease on day 5 ( ig/g tissue) and remained at this level until day 7. On day 8 the histamine on- tent fell to tg/g tissue whih was similar to values found in normal skin. Mediator ontent of homografts Homografts of leg skin transplanted onto the baks of 4 rabbits were removed individually at intervals from day 2 to day 7 after grafting. The different mediator ontents assayed in the homograft tissue are illustrated in Figure 3. As with the autografts, during the initial healing-in period, the ontents of all mediators were similar to those of normal skin exept for histamine. There was no hange in the 5-HT ontent throughout the ourse of the experiment. The histamine ontent reahed two peaks, one on day 4, hour 12 ( tg/g tissue) and the seond on day 5, hour 12 ( tg/g tissue) i.e. after the blood flow had stopped ompletely. Between these peaks, on day 5, the histamine ontent fell signifiantly to jg/g tissue. By day 6 the histamine ontent had dereased and ontinued to do so up to day 7 ( jg/g tissue). The hanges in prostaglandin ontent produed a different piture from that of the histamine ontent. The PGE2 remained low up to day 4 but between day 4, hour 12 and day 5 the ontent inreased ( ng/g tissue), forming a short-lived plateau whih oinided with the peak of blood flow. After

4 126 G.P. LEWS & BEVERLEY A. MANGHAM E) b (a a ndomethain (mg kg-' day-1) Figure 4 The effet of indomethain on the blood flow hanges in rabbit skin homografts. Blood flow is expressed as in Figure 1. Two groups of animals reeived indomethain 2 x 5 mg kg-1 day-' (A) and 2 x 1 mg kg-' day-' (A) given intravenously from day 3 after grafting. Eah point represents the mean; vertial lines show s.e. means. The homografts on animals treated with indomethain failed to reah the maximum blood flow of the untreated homografts (). This is more learly illustrated in (b) by the histograms whih represent the mean of the peak flow attained in eah animal. The effet of indomethain was to derease the maximum blood flow by 35-4%. The numbers in the histograms refer to the number of animals. the blood flow in the homograft had stopped there was a further inrease to very high levels up to day 7 ( ng/g tissue) after grafting. At the time of the main inrease in PGE2 ontent there was also an inrease in PGF2e ontent. On day 5, hour 12 this reahed ng/g tissue. Effet of antagonists on the blood flow n a series of experiments in whih animals reeiving autografts were treated with indomethain, mepyramine, metiamide or methysergide before grafting, there was no influene on the blood flow pattern established by the autografts. On the other hand, both indomethain and mepyramine affeted the blood flow hanges during the homograft reation. Two groups of 6 animals were given indomethain (5 and 1 mg/kg) intravenously twie a day from day 3 after grafting. n neither group did the blood flow reah the maximum blood flow found in the untreated homografts (Figure 4). The effet of indomethain on the maximum blood flow J u o o Figure 5 The effet of mepyramine on the blood flow hanges in rabbit skin homografts. Blood flow is expressed as in Figure 1. Two groups of animals reeived mepyramine 3 x 2.5 mg kg-' day-', group (A) from day 3 and group 11 {A) from day 1 after grafting until rejetion had ourred. The essation of blood flow of the homografts of the mepyramine-treated animals ourred 7 to 1 days after grafting ompared with day 5 in the untreated homografts (@). Eah point represents the mean of results from 4 to 7 animals; vertial lines show s.e. means. ahieved by the individual homografts is further illustrated in Figure 4b. Although indomethain affeted the maximum blood flow, it did not alter the time of rejetion and hene the survival of the homograft. While metiamide (3 x 5 mg kg-' day-') (H2-reeptor antagonist) did not alter the blood flow pattern, mepyramine (H-reeptor antagonist) prolonged the inreased blood flow and inreased the homograft survival time from 5 days to 7 to 1 days (Figure 5). Two groups of animals were treated with mepyramine (3 x 2.5 mg kg-1 day-') group (5 animals) from day 3 and group (4 animals) from day 1 after grafting until rejetion had ourred. The time at whih mepyramine treatment began did not signifiantly alter the effet on the blood flow of the homograft. When three animals were treated with a ombination of mepyramine and metiamide from day 1, the peak of high blood flow in the homograft was maintained for about 36 h instead of 12 h in untreated animals before rejetion ourred. Two groups of animals reeived methysergide (5-HT antagonist) either from day 1 or day 3 after grafting until rejetion had ourred. Methysergide like metiamide, did not alter the blood flow pattern established by the homograft.

5 BLOOD FLOW AND MEDATORS N GRAFTS j2 (a 1- o m) 3- a ') l) ol a : -- :45r_ 91'6 El ' b > o E N,) D 15- L ) - -. r3 m t b Figure 6 omparison of blood flow hanges () with the ontent of histamine (o) and prostaglandin E2 (PGE2, A) in the rabbit skin autograft ( and homograft (b). Data were obtained from Figures 1, 2 and 3; blood flow is expressed as in Figure 1. There is a good orrelation between the blood flow hanges and the histamine ontent during the healing-in proess in both types of grafts. The peak flow in homografts was aompanied by a small signifiant inrease in PGE2 ontent. The essation of blood flow in homografts ours at a time when the histamine ontent was maximal. The seondary inrease in the histamine and PGE2 ontents ourred after rejetion of the homograft i.e. essation of blood flow. Eah point represents the mean; vertial lines show s.e. means. Disussion The purpose of this study was to investigate the possible ontribution made by pharmaologial mediators to the hanges of blood flow whih our in skin autografts and homografts. Although the hanges have been well doumented by visual observations, in the present study 133Xe learane has been used as a more preise measure of blood flow. When transplanted skin beomes revasularised there is a pronouned vasodilatation in the tissue and in the ase of the homograft vasoonstrition ours at the onset of rejetion. n the present experiments the inreased n a1) 3 vasodilatation was maintained for several days in the autografts until the flow returned to that of the normal surrounding skin. n homografts the peak flow was maintained for only about 12 h until the onset of rejetion when the blood flow eased. nrease in blood flow during the healing-in of both autografts and homografts was aompanied by an inrease in tissue histamine (see Figure 6). However, if the histamine present in the tissue is responsible for produing this vasodilatation, it would appear to be released in a form that is not aessible to antagonists sine neither H1- nor H2-histamine reeptor antagonists, when given systemially throughout the experiment, altered the blood flow hanges during the healing-in proess. The taking of a graft involves growth of new tissue and is similar to the tissue repair proess studied by other workers (Kahlson, 1962; Moore & Shayer, 1969). Kahlson and his olleagues (1962) in studying the proess of tissue growth have suggested that histamine is formed by the ation of the enzyme histidine dearboxylase as 'nasent' histamine whih is not antagonized by antihistamines. Shayer (1962) has shown that this enzyme has an induible ativity and he has proposed an important role for histamine in miroirulatory regulation. The indued histamine may be synthesized in or near vasular endothelial ells and may be intrinsi aording to the definition of Dale (1948). n the present experiments, therefore, the tissue histamine found during the healing-in of autografts and homografts represent 'nasent' or intrinsi histamine, whih would explain the resistane of the vasular effet to antihistamine agents. However, as tissue homogenates were used for extration of the mediators, it was not possible to distinguish between bound and free histamine. Therefore, during the healing-in of the grafts it is possible that the histamine formed remained bound or intraellular so that it was not funtional and therefore not responsible for the onomitant vasodilatation. Both H1- and H2-histamine reeptors have been shown to exist in the peripheral vasular system of the rabbit (Parsons & Owen, 1973; Brimbleombe, Owen & Parsons, 1974). These workers have suggested that the normal response to histamine reflets the balane between the opposing vasoonstritor (H1-reeptors) and the vasodilator (H2-reeptors) effets. This balane hanges under differing onditions. The vasodilator phase usually predominates when smaller doses of histamine are given while larger doses of histamine give rise to prolonged vasoonstrition. The histamine ontent of the homograft at its highest i.e. just before essation of blood flow (see Figure 6) was at least 3 times greater than that in the autograft and more than 6 times that in ontrol skin. t seems possible that whereas the onentration of hist-

6 128 G.P. LEWS & BEVERLEY A. MANGHAM amine present during the healing-in proess was only suffiient to ause vasodilatation, at its peak onentration the histamine aused vasoonstrition suffiient to bring on the onset of rejetion of the tissue. Unlike the histamine present during the early stages of the reation, the histamine present during the peak onentration appeared to at on Hl-reeptors to ause the vasoonstrition beause in animals treated with the antagonist, mepyramine, the vasoonstrition was overome and the onset of rejetion was delayed from 5 days up to 7 to 1 days. The animals were treated with the antihistamine throughout the experiment until rejetion ourred. n spite of this, delayed rejetion still ourred, aompanied by essation of blood flow. t is not lear at present whether suffiient histamine was generated to overome the. effet of mepyramine or some other vasoonstritor mehanism was involved. Henney, Bourne & Lihtenstein (1972) suggested that histamine has the ability to suppress the ytolyti ativity of T-lymphoytes and that this ation is mediated via an H2-reeptor. t is possible that mepyramine being an H-antagonist ats by diverting the limited amount of histamine available preferentially to H2-reeptor sites on the sensitized lymphoytes, and onsequently delaying rejetion. However, it was not possible to test this view by the use of the H2-reeptor antagonists in the present investigation, sine the rejetion reation ourred at the maximum intensity and any potentiation that might have ourred with H2-reeptor antagonists would not have been evident. However, it was evident that the delayed rejetion aused by mepyramine was partly reversed by metiamide. Sine indomethain, a potent inhibitor of prostaglandin formation, did not delay the onset of rejetion, it appears that prostaglandins do not play a role in this part of the reation. However, it might well be that a prostaglandin of the E series, probably E2, is partly responsible for the peak inrease of blood flow in homografts. Firstly, at this time there was a small but signifiant inrease in the PGE2 ontent of the homografts but not the autografts. Seondly, the homografts on animals treated with indomethain all failed to reah the peak blood flow of the untreated homografts; this was redued by 35-4%. The peak blood flow in autografts was unaffeted by indomethain. This work was supported by the MR, the Vandervell Foundation and the Smith & Nephew Foundation. Referenes BOGDANSK, D.F., PLETSHER, A., BRODE, B.B. & UDEN- FREND, S. (1956) dentifiation and assay of serotonin in brain. J. Pharma. exp. Ther., 117, BRMBLEOMBE, R.W., OWEN, D.A.A., & PARSONS, M.E. (1974) The ardiovasular effets of histamine in laboratory animals. Agents and Ations, 4, DALE, H. (1948) Antihistamine substanes. Br. med. J. 2, EVANS, D.P., LEWS, J.A. & THOMSON, D.S. (1973) An automated fluorimetri assay for the rapid determination of histamine in biologial fluids. Life Si., 12, GLMORE, N., VANE, J.R. & WYLLE, J.H. (1968) Prostaglandins released by the spleen. Nature, 218, HENNAM, J.R., JOHNSON, D.A., NEWTON, J.R. & OLLNS, W.P. (1974) Radioimmunoassay of prostaglandin F2 in peripheral venous plasma from men and women. Prostaglandins, 5, HENNEY,.S., BOURNE, H.R., LHTENSTEN, L.M. (1972) The role of yli 3'5'adenosine monophosphate in the speifi ativity of lymphoytes. J. mmunol., 18, JASAN, M.K. & LEWS, G.P. (1971) Lymph flow and hanges in intraellular enzymes during healing and rejetion of rabbit skin grafts. J. Physiol., 219, KAHLSON, G. (1962) New approahes to the physiology of histamine. Perspetives Biol. Med., 5, LEWS, G.P., PEK, M.J., WLLAMS, T.J. & YOUNG, B.A. (1976) Measurement of blood flow in rabbit skin homografts and autografts using a '33Xenon learane tehnique. J. Physiol., 254, 32-33P. MAKEL, R.P. & MLLER, F.P. (1966) Fluoresent produts formed by reation of indole derivatives and o-phthalaldehyde. Anal. hem., 38, MEDAWAR, P.B. (1944) The behaviour and fate of skin autografts and skin homografts in. rabbits. J. Anat., 78, MOORE, T.. & SHAYER, R.W. (1969) Histidine dearboxylase ativity of autografted and allografted rat skin. Transplantation, 7, PARSONS, M.E., & OWEN, D.A.A. (1973) Reeptors involved in the ardiovasular responses to histamine. n Pro. nt. Symp. on Histamine H2-Reeptor Antagonists. ed. Wood,.J. & Simkins, M.A. pp Welwyn Garden ity: Smith, Kline & Frenh. SHAYER, R.W. (1962) Evidene that indued histamine is an intrinsi regulator of the miroirulatory system. Am. J. Physiol., 22, SEJRSEN, P. (1969) Blood flow in utaneous tissue in man studied by washout of radioative Xenon. irulation Res., 25, SHORE, P.A., BURKHALTER, A. & OHEN, V.H. (1959) A method for the fluorometri assay of histamine in tissues. J. Pharma. exp. Ther., 127, (Reeived February 28, Revised April 12, 1978.)