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The microvasculature of the spleen
Clin. RadioL (1976) 27, 259-264 THE MICROVASCULATURE
OF THE SPLEEN
A. B. AYERS,* t K. HENRY,:~ S. B. RUSSELL and R. E. STEINER
From the Department of Diagnostic Radiology and Department of Pathology, Hammersmith Hospital, Du Cane Road, London W.12 A method for examining the microvasculature of the dog spleen by angiography is described and the findings are related to morphological studies. The marginal sinus of the lymphoid follicle has been shown to be an important part of the vascular pathway in the spleen. It allows intimate mixing of blood elements and spleen cells and it is suggested that this plays an important immunological role. The control of blood flow to the lymphoid follicle is discussed but requires further elucidation.
butal, Abbott Laboratories Ltd). Four dogs were premedicated, one hour before i.v. anaesthesia, with 50 mg i.m. of diethylthiambutene hydrochloride (Themalon, Burroughs Wellcome). Tracheal intubation was performed with an 11 mm Magill cuffed endotracheal tube and the bladder was catheterised. Systemic blood pressure was measured throughout via a left femoral artery catheter, using a Statham high pressure transducer (P23Db) and was continuously displayed on an Electronics for Medicine oscilloscope and recorded by an ElemaSchonander 81 Mingograf. A midline abdominal incision was made and spleen dissected free of its attachments, apart from the splenic artery, vein and post-ganglionic sympathetic never trunk, as described by Davies and Withrington (1968). Splenic arterial blood flow was continuously measured with an electromagnetic cuff flow probe (size 4 mm) and flow meter (S.E. Laboratories). In nine experiments the nerve trunk was dissected free from the artery and divided. The distal end was placed on platinum stimulating METHODS AND MATERIALS electrodes with the cathode peripheral for stimulaExperiments were performed on 25 mongrel dogs tion with a CF Palmer 8044 stimulator. weighing between 12.5 and 30 kg. These were Splenic arterial catheters were introduced via a anaesthetised with an intravenous injection of right femoral arteriotomy. Whenever possible a fine 6-8 ml of 5% thiopentone sodium (Pentothal, white Portex catheter was manipulated into a branch Abbott Laboratories Ltd) followed by intermittent of the splenic artery but on the few occasions when doses of 2 ml of 6 % pentobarbitone sodium (Nero- this was not possible a Judkin's visceral catheter was positioned in the main splenic artery. The spleen was then exteriorised via the abdomi*This article is based on the paper read at the 1974Exeter meetingof the Faculty of Radiologistsfor whichthe Couch nal incision and placed on a perspex handbuilt film Award was received. changer holding a maximum of five films (Industrex A, Kodak). Sequential films were exposed after the tPresent address: St. Thomas's Hospital, London. injection of 8-10 ml of Urografin 6070 (Schering) ~Present address: WestminsterHospital, London.
THB exact arteriovenous pathway in the spleen has been debated for many years, but the anatomy of the main arteries, venous sinuses and trabecular veins is well established. The venous sinuses have attracted much attention as a site of blood storage in certain animal species and have been well described (Krumbaar, 1926; McNee, 1931; Knisely, 1936; Bjorkman, 1947; Snook, 1950; Lewis, 1957). The arteriolar and capillary anatomy has also been described in detail (Robinson, 1926: MacNeal et al., 1927; MacNeal, 1929; Nisimaru and Steggerda, 1932) but debate still exists about the exact pathway and its functional significance. There are some difficulties in comparing animal and human studies because of the species variation that is well recognised. The purpose of this paper is to report the findings of an investigation of the microvasculature of the dog's spleen using radiologic and injection methods and to briefly discuss these findings in relation to the spleen's immunological role.
259
260
CLINICAL
using a focal spot size of 0-26 mm (Siemens) and a focal film distance of 40 cm. Injection was made by a mechanical pump using 6 kg weight. Exposure factors were 55 KV and 8-12 mAs with an exposure time of 0.06-0.08 s. The films were developed by a Pakorol 12min automatic cycle using G138 Developer (Agfa Gevaert), at 75°C. On occasions drugs were injected via the splenic catheter before contrast injection. The following drugs were used: noradrenaline tartrate (Levophed, Winthrop), adrenaline tartrate, BP, isoprenaline sulphate BP, angiotensin amide (Hypertensin, Ciba) and vasopressin (Pitressin, Parke Davis & Co). P o s t - m o r t e m Injections. - Five spleens were removed after the other experimental studies and injected either by arterial or venous route with microfil (Canton Bio-medical Products, Inc.) monitoring the injection pressure in order to avoid exceeding the normal arterial or venous pressures accordingly. These specimens were fixed in formal saline for at least two days before 2-4 mm slices were cut. These were dehydrated for 24 h in each increasing concentration of ethyl alcohol, and finally cleared with methyl salicyclate BP (Evans Medical). Three further spleens were removed after an injection into the splenic artery of 2 ml Indian ink in vivo, which was allowed to circulate for 10 min. Two spleens were injected post-mortem with intraarterial barium sulphate.
RESULTS A. Angiography. - Angiography has provided a dynamic method of demonstrating the passage of blood through the spleen (Figs. 1-4). The major trabecular arteries and branches could be visualised at first but also seen at an early stage were numerous haloes of contrast closely connected to the smaller splenic arteries. These were distributed throughout the spleen and appeared in each animal to be fairly uniform in size. Each interlobular artery was seen to supply a group of haloes within its territory. The number of haloes supplied depended on the size of the territory. Anastomoses between the interlobular arteries were not demonstrated. The opacification of the haloes was seen in both arterial and venous phases (Figs. 1, 3) and some of the contrast was seen in these areas even in the late venous phase, some 14 s after injection (Fig. 4). At this stage of the study the splenic pulp was also more generally opacified but the outer margins of the haloes were difficult to define.
RADIOLOGY
By photographic magnification the haloes could be seen more distinctly and their size determined (Fig. 5). The mean width of the central lucency was constant in all phases of arteriography and measured 360 ~m +_24. This area appeared not to be opacify to any real degree in any phase of arteriography. The width of the opacified haloes was measured during arterial and venous phases. The latter measurement was often difficult since during the venous phases the outer margin of the halo became less distinct and was difficult to differentiate from the general opacification occurring in the spleen at this time. The mean width during the arterial phase was 130 ~m + 12, whereas during the venous phase it was 230 [xm + 16. The inner lucent zone remained a constant size throughout all phases of angiography and the increase in halo width could be seen to be caused by contrast spreading centrifugally. The exact connections between the finer arterioles, the haloes, the venous sinuses and the veins have not been shown by angiography but the appearances suggest that contrast passed directly from the arteries to the haloes and thence diffused over a period of 8-14 s into the venous sinuses and to the draining veins. B. Carbon Injections. - It has been known for a long time that foreign particulate matter injected into the splenic circulation becomes localised in the marginal zone (MacNeal et al., 1927). In the present experiments 2 ml of Indian ink were injected into the splenic artery 10 rain before removal. Fig. 6 shows the typical appearances of a lymphoid follicle with a surrounding band of intra- and extraeellular carbon particles in the marginal zone including the marginal sinus. The lymphoid follicle outlined by carbon coincided in size to the lucent zones seen on the arteriographs. C. B a r i u m Sulphate Injections. - Post-mortem injections of barium sulphate were made on two occasions and radiographs of spleen slices revealed haloes similar to those seen at angiography. Microscopic examination of these showed that the barium was lying in the marginal zone around the lymphoid follicle (Malpighian corpuscle) of the spleen. These studies confirmed the angiographic and morphological findings. D . Microfil Injections. - Microfil was injected arterially or retrogradely via the splenic vein or both simultaneously.
ARTERIALPATHWAYS.- By injecting small volumes intra-arterially we were able to demonstrate the
THE M I C R O V A S C U L A T U R E OF THE SPLEEN
FIGS. 1-4 Angiograms of the spleen taken at 2, 4, 8 and 14 s after injection of" contrast. A n omental artery has also been opacified.
261
262
CLINICAL RADIOLOGY
FIG. 7 An arterial m i c r o n injection ( x 80) showing the arterial supply to a lymphoid follicle. A---interlobular artery; al, a2 and a3 = arterioles; MS = marginal sinus.
FIG. 5 A magnified print of an angiogram taken at 4 s after injection of contrast. A 1 m m marker is shown.
FIG. 8 A venous microfil injection ( x 15) showing the venous sinus and trabecula veins. Note the empty spaces for the lymphoid follicles.
FIG. 6 A photomicrograph of a lymphoid follicle ( x 250) after Indian ink injection, its distribution in the marginal zone showing.
finer vascular connections between the arteries and the marginal sinuses (Fig. 7). The marginal sinuses were filled in three ways. Firstly by small arteries arising from the interlobular artery before the lymphoid follicle and entering the outer aspect of
the sinus. Secondly by arterioles arising from the central artery of the follicle traversing the lymphoid tissue and entering the inner aspect of the marginal sinus. Thirdly by arteries arising from the central artery after leaving the follicle and supplying the outer aspect of the marginal sinus. Injected retrogradely the trabecula veins and venous sinuses were demonstrated (Fig. 8). The marginal sinuses were not filled by this method of injection nor was the arterial tree. Combined injections have demonstrated the relationship of the
THE
MICROVASCULATURE
OF
THE
lymphoid follicle including the marginal sinus surrounded by the smaller venous sinuses, which then drain into the splenic veins.
E. The Effects of Physiological stimuli. - Nerve stimulation at high physiological frequencies (5-10 Hz) causes reduction in blood flow by vasoconstriction and a reduction in spleen volume by capsular and trabecular smooth muscle contraction which disturbed the angiographic pattern and the marginal zones became indistinct. A similar pattern was found after the intra-arterial injection of adrenaline and noradrenaline (5 ~Lg). However at lower frequencies of nerve stimulation (0.5-1 Hz) and at lower doses of these catecholamines (0.5 sg), when capsular contraction is known to be less marked (Davies et al., 1968, 1973), the marginal zones were seen to be filled with contrast in a normal manner. Vasoconstriction can also be obtained by the intra-arterial injections of angiotensin (0.1-1.0 ~g) and vasopressin (0.5-5.0i.u.) without significant capsular response (Ayers et al., 1972). Angiography performed during the action of these drugs demonstrated that the marginal sinus filled with contrast for a considerably longer time, sometimes up to 25 s. DISCUSSION MacNeal et al. (1927) studied the circulation of human and animal spleens using a technique of fixation of the spleen in a distended state and so were able to identify the circulatory pathways. These authors concluded that these vascular pathways enabled an intimate contact between the formed elements of the blood and the reticuloendothelial cells of the marginal zone. The anglographic studies reported here have demonstrated the importance of the marginal zone as a major blood pathway. Interest in the marginal zone has also been aroused in recent years following the demonstration of rapid localisation of injected antigens in the perifollicular region (Nossal et al., 1966; Mitchell and Abbott, 1971). Mitchell (1972) also demonstrated the migration of antigen-binding cells, prepared in vitro and injected in vivo, from the marginal zone to the germinal centre. Angiography has demonstrated that the marginal zones remain filled with contrast for a considerable period of time, haemodynamically. This delay in passage of the contrast media would provide a mechanism whereby there is prolonged contact of blood and particulate matter including antigen with the reticulo-endothelial cells of the marginal zone. Thus the perifollicular mononuclear phagocytes are
263
SPLEEN
-
•
a3
1_
M.s.
.......:.~ "~'~(,~.
':)":;":":'": :!':::
~
~ ~::
L.F. ".!....'
"V. V Fro. 9 An artist's impression of the splenic microvasculature, A, interlobular artery; al, a2 and a3, arterioles; MS, marginal sinus; CA, central arteriole; LF, lymphoid follicle; VS, venous sinus; V, vein. able to selectively assess particulate matter and cells in the blood for antigen processing or phagocytosis. MacNeal (1929) described two types of arteriole supplying the marginal sinus, namely, a branch of the interlobular artery arising before the lymphoid follicle and the terminations of the follicular capillaries. Our microfil studies have demonstrated a further arteriole supplying the marginal sinus which arises from the interlobular artery after it has passed through a lymphoid follicle. Whether these arteriolar branches of the interlobular arteries (al and as, Figs. 7, 9) correspond to the penicillar arteries described by many other authors is debatable. It has long been stated that the splenic arteries are end arteries and that there are no anastomoses between each lobule. Our angiographic studies have failed to show any anastomotic channels during normal flow or during physiological stimuli. However it has not been recognised previously that each follicular artery supplies a number of Malpighian bodies and this has been clearly shown by arteriography. MacNeal also suggested that these arterioles functioned intermittently similar to those of the skin (Krogh, 1921) and those of the renal capillary tufts (Richards, 1925). If perfusion of a single marginal zone was intermittent it would provide a mechanism whereby blood could be held in the sinus for a considerable period of time and thus explain the angiographic findings. The control of the mechanism has still to be elucidated.
264
CLINICAL RADIOLOGY
I f the b l o o d flow to the spleen is reduced by nerve stimulation at a high physiological frequency (5-10 Hz) or by intra-arterial injection of adrenaline or n o r a d r e n a l i n e (2-5-5 ~zg) the opacification o f the marginal zone at a n g i o g r a p h y can be disturbed. However, this m a y be due to the contraction of the smooth muscle in the trabeculae a n d capsule of the spleen rather t h a n alterations in the vascular pathway, because when similar vascular changes were obtained b y injecting drugs which do n o t have significant capsular activity such as Vasopressin a n d Angiotensin the m a r g i n a l sinuses are seen to fill clearly. I n the latter case the only change observed was that the sinuses r e m a i n e d full of contrast for a more prolonged period, sometimes u p to 25 s. Acknowledgements. - We are indebted to Mr W. Hinkes for his photographic skill, and to Miss F. Cuthill for the drawing. REFERENCES AYERS, A. B., DAVmS,B. N. & WITrmIN~TON,P. G. (1972). Responses of the isolated perfused human spleen to sympathetic nerve stimulation, catecholamines and polypeptides. British Journal o f Pharmacology, 44, 17-30. BJORKMAN,S. E. (1947). The splenic circulation with special reference to the function of the spleen sinus wall. Acta medica scandinavica, Supplementa 191. DAVIES, B. N., GAMBLE,J. & WIa~RIN~TON,P. G. (1968). Effects of noradrenaline, adrenaline and angiotensin on the vascular and capsular smooth muscle of the spleen of the dog. British Journal of Pharmacology and Chemotherapy, 32, 424P. DAWES, B. N., GAMBLE,J. & WITHRINGTON,P. G. (1973). Frequency-dependent differences in the responses of the capsular and vascular smooth muscle of the spleen of the dog to sympathetic nerve stimulation.Journal of Physiology (London), 228, 13-25. DAwEs, B. N. & WITHRIN~TON,P. G. (1968). The effects of
prostaglandin E1 and E2 on the smooth muscle of the dog spleen and on its response to catecholamines, angiotensin and nerve stimulation. British Journal of Pharmacology and Chemotherapy, 32, 136-144. KNISELY,M. H. (1936). Spleen studies: microscopic observation of the circulatory system of living unstimulated mammalian spleens. Anatomical Record, 65, 131-148. KROGn, A. (1921). Studies on the physiology of capillaries. II. The reactions to local stimuli of the blood vessels in the skin and web of the frog. Journal of Physiology (London), 55, 41~422. KRUMBrIAAR,E. B. (1926). Functions of the spleen. Physiological Reviews, VI, 160-200. LEwis, O. J. (1957). The blood vessels of the adult mammalian spleen. Journal of Anatomy, 91,245-250. MACNEAL,W. J. (1929). The circulation of blood through the spleen pulp. Archives of Pathology, 7, 215-227. MACNEAL, W. J., OTANI, S. & PATTERSON,M. B. (1927). The finer vascular channels of the spleen. American Journal of Pathology, 3, 111-112. McNEE J. W. (1931). The spleen: its structure function and diseases. Lancet, 220, 951-957, 1009-1015, 1063-1070. MITCHELL, J. (1972). Antigens in immunity. XVIII. The migration of antigen-binding, bone-marrow-derived and thymus derived spleen cells in mice. immunology, 22, 231-245. MITCHELL,J. & ABBOT,A. (1971). Antigens in immunity.XVI. A light and electron microscope study of antigen localisation in the rat spleen. Immunology, 21, 207-224. NISIMARU,Y. • STEGGARDA,F. R. (1932). Observations on the structure and function of certain blood vessels in the spleen. Journal of Physiology (London), 74, 327-337. NOSSAL, G. J. V., AUSTIN, C. M., PYE, J. & MITCHELL,J. (1966). Antigens in immunity. XII. Antigen trapping in the spleen. International Archives of Allergy and Applied Immunology, 29, 368-383. RICHARDS,A. N. (1925). The nature and mode of regulations of glomerular function. American Journal of Medical Science, 170, 781-803. ROBINSON, W. L. (1926). Vascular mechanism of the spleen. American Journal of Pathology, 2, 341-355. SNOOK, T. (1950). A comparative study of the vascular compartments in mammalian spleens. American Journal of Anatomy, 87, 31-77.
OF THE SPLEEN
A. B. AYERS,* t K. HENRY,:~ S. B. RUSSELL and R. E. STEINER
From the Department of Diagnostic Radiology and Department of Pathology, Hammersmith Hospital, Du Cane Road, London W.12 A method for examining the microvasculature of the dog spleen by angiography is described and the findings are related to morphological studies. The marginal sinus of the lymphoid follicle has been shown to be an important part of the vascular pathway in the spleen. It allows intimate mixing of blood elements and spleen cells and it is suggested that this plays an important immunological role. The control of blood flow to the lymphoid follicle is discussed but requires further elucidation.
butal, Abbott Laboratories Ltd). Four dogs were premedicated, one hour before i.v. anaesthesia, with 50 mg i.m. of diethylthiambutene hydrochloride (Themalon, Burroughs Wellcome). Tracheal intubation was performed with an 11 mm Magill cuffed endotracheal tube and the bladder was catheterised. Systemic blood pressure was measured throughout via a left femoral artery catheter, using a Statham high pressure transducer (P23Db) and was continuously displayed on an Electronics for Medicine oscilloscope and recorded by an ElemaSchonander 81 Mingograf. A midline abdominal incision was made and spleen dissected free of its attachments, apart from the splenic artery, vein and post-ganglionic sympathetic never trunk, as described by Davies and Withrington (1968). Splenic arterial blood flow was continuously measured with an electromagnetic cuff flow probe (size 4 mm) and flow meter (S.E. Laboratories). In nine experiments the nerve trunk was dissected free from the artery and divided. The distal end was placed on platinum stimulating METHODS AND MATERIALS electrodes with the cathode peripheral for stimulaExperiments were performed on 25 mongrel dogs tion with a CF Palmer 8044 stimulator. weighing between 12.5 and 30 kg. These were Splenic arterial catheters were introduced via a anaesthetised with an intravenous injection of right femoral arteriotomy. Whenever possible a fine 6-8 ml of 5% thiopentone sodium (Pentothal, white Portex catheter was manipulated into a branch Abbott Laboratories Ltd) followed by intermittent of the splenic artery but on the few occasions when doses of 2 ml of 6 % pentobarbitone sodium (Nero- this was not possible a Judkin's visceral catheter was positioned in the main splenic artery. The spleen was then exteriorised via the abdomi*This article is based on the paper read at the 1974Exeter meetingof the Faculty of Radiologistsfor whichthe Couch nal incision and placed on a perspex handbuilt film Award was received. changer holding a maximum of five films (Industrex A, Kodak). Sequential films were exposed after the tPresent address: St. Thomas's Hospital, London. injection of 8-10 ml of Urografin 6070 (Schering) ~Present address: WestminsterHospital, London.
THB exact arteriovenous pathway in the spleen has been debated for many years, but the anatomy of the main arteries, venous sinuses and trabecular veins is well established. The venous sinuses have attracted much attention as a site of blood storage in certain animal species and have been well described (Krumbaar, 1926; McNee, 1931; Knisely, 1936; Bjorkman, 1947; Snook, 1950; Lewis, 1957). The arteriolar and capillary anatomy has also been described in detail (Robinson, 1926: MacNeal et al., 1927; MacNeal, 1929; Nisimaru and Steggerda, 1932) but debate still exists about the exact pathway and its functional significance. There are some difficulties in comparing animal and human studies because of the species variation that is well recognised. The purpose of this paper is to report the findings of an investigation of the microvasculature of the dog's spleen using radiologic and injection methods and to briefly discuss these findings in relation to the spleen's immunological role.
259
260
CLINICAL
using a focal spot size of 0-26 mm (Siemens) and a focal film distance of 40 cm. Injection was made by a mechanical pump using 6 kg weight. Exposure factors were 55 KV and 8-12 mAs with an exposure time of 0.06-0.08 s. The films were developed by a Pakorol 12min automatic cycle using G138 Developer (Agfa Gevaert), at 75°C. On occasions drugs were injected via the splenic catheter before contrast injection. The following drugs were used: noradrenaline tartrate (Levophed, Winthrop), adrenaline tartrate, BP, isoprenaline sulphate BP, angiotensin amide (Hypertensin, Ciba) and vasopressin (Pitressin, Parke Davis & Co). P o s t - m o r t e m Injections. - Five spleens were removed after the other experimental studies and injected either by arterial or venous route with microfil (Canton Bio-medical Products, Inc.) monitoring the injection pressure in order to avoid exceeding the normal arterial or venous pressures accordingly. These specimens were fixed in formal saline for at least two days before 2-4 mm slices were cut. These were dehydrated for 24 h in each increasing concentration of ethyl alcohol, and finally cleared with methyl salicyclate BP (Evans Medical). Three further spleens were removed after an injection into the splenic artery of 2 ml Indian ink in vivo, which was allowed to circulate for 10 min. Two spleens were injected post-mortem with intraarterial barium sulphate.
RESULTS A. Angiography. - Angiography has provided a dynamic method of demonstrating the passage of blood through the spleen (Figs. 1-4). The major trabecular arteries and branches could be visualised at first but also seen at an early stage were numerous haloes of contrast closely connected to the smaller splenic arteries. These were distributed throughout the spleen and appeared in each animal to be fairly uniform in size. Each interlobular artery was seen to supply a group of haloes within its territory. The number of haloes supplied depended on the size of the territory. Anastomoses between the interlobular arteries were not demonstrated. The opacification of the haloes was seen in both arterial and venous phases (Figs. 1, 3) and some of the contrast was seen in these areas even in the late venous phase, some 14 s after injection (Fig. 4). At this stage of the study the splenic pulp was also more generally opacified but the outer margins of the haloes were difficult to define.
RADIOLOGY
By photographic magnification the haloes could be seen more distinctly and their size determined (Fig. 5). The mean width of the central lucency was constant in all phases of arteriography and measured 360 ~m +_24. This area appeared not to be opacify to any real degree in any phase of arteriography. The width of the opacified haloes was measured during arterial and venous phases. The latter measurement was often difficult since during the venous phases the outer margin of the halo became less distinct and was difficult to differentiate from the general opacification occurring in the spleen at this time. The mean width during the arterial phase was 130 ~m + 12, whereas during the venous phase it was 230 [xm + 16. The inner lucent zone remained a constant size throughout all phases of angiography and the increase in halo width could be seen to be caused by contrast spreading centrifugally. The exact connections between the finer arterioles, the haloes, the venous sinuses and the veins have not been shown by angiography but the appearances suggest that contrast passed directly from the arteries to the haloes and thence diffused over a period of 8-14 s into the venous sinuses and to the draining veins. B. Carbon Injections. - It has been known for a long time that foreign particulate matter injected into the splenic circulation becomes localised in the marginal zone (MacNeal et al., 1927). In the present experiments 2 ml of Indian ink were injected into the splenic artery 10 rain before removal. Fig. 6 shows the typical appearances of a lymphoid follicle with a surrounding band of intra- and extraeellular carbon particles in the marginal zone including the marginal sinus. The lymphoid follicle outlined by carbon coincided in size to the lucent zones seen on the arteriographs. C. B a r i u m Sulphate Injections. - Post-mortem injections of barium sulphate were made on two occasions and radiographs of spleen slices revealed haloes similar to those seen at angiography. Microscopic examination of these showed that the barium was lying in the marginal zone around the lymphoid follicle (Malpighian corpuscle) of the spleen. These studies confirmed the angiographic and morphological findings. D . Microfil Injections. - Microfil was injected arterially or retrogradely via the splenic vein or both simultaneously.
ARTERIALPATHWAYS.- By injecting small volumes intra-arterially we were able to demonstrate the
THE M I C R O V A S C U L A T U R E OF THE SPLEEN
FIGS. 1-4 Angiograms of the spleen taken at 2, 4, 8 and 14 s after injection of" contrast. A n omental artery has also been opacified.
261
262
CLINICAL RADIOLOGY
FIG. 7 An arterial m i c r o n injection ( x 80) showing the arterial supply to a lymphoid follicle. A---interlobular artery; al, a2 and a3 = arterioles; MS = marginal sinus.
FIG. 5 A magnified print of an angiogram taken at 4 s after injection of contrast. A 1 m m marker is shown.
FIG. 8 A venous microfil injection ( x 15) showing the venous sinus and trabecula veins. Note the empty spaces for the lymphoid follicles.
FIG. 6 A photomicrograph of a lymphoid follicle ( x 250) after Indian ink injection, its distribution in the marginal zone showing.
finer vascular connections between the arteries and the marginal sinuses (Fig. 7). The marginal sinuses were filled in three ways. Firstly by small arteries arising from the interlobular artery before the lymphoid follicle and entering the outer aspect of
the sinus. Secondly by arterioles arising from the central artery of the follicle traversing the lymphoid tissue and entering the inner aspect of the marginal sinus. Thirdly by arteries arising from the central artery after leaving the follicle and supplying the outer aspect of the marginal sinus. Injected retrogradely the trabecula veins and venous sinuses were demonstrated (Fig. 8). The marginal sinuses were not filled by this method of injection nor was the arterial tree. Combined injections have demonstrated the relationship of the
THE
MICROVASCULATURE
OF
THE
lymphoid follicle including the marginal sinus surrounded by the smaller venous sinuses, which then drain into the splenic veins.
E. The Effects of Physiological stimuli. - Nerve stimulation at high physiological frequencies (5-10 Hz) causes reduction in blood flow by vasoconstriction and a reduction in spleen volume by capsular and trabecular smooth muscle contraction which disturbed the angiographic pattern and the marginal zones became indistinct. A similar pattern was found after the intra-arterial injection of adrenaline and noradrenaline (5 ~Lg). However at lower frequencies of nerve stimulation (0.5-1 Hz) and at lower doses of these catecholamines (0.5 sg), when capsular contraction is known to be less marked (Davies et al., 1968, 1973), the marginal zones were seen to be filled with contrast in a normal manner. Vasoconstriction can also be obtained by the intra-arterial injections of angiotensin (0.1-1.0 ~g) and vasopressin (0.5-5.0i.u.) without significant capsular response (Ayers et al., 1972). Angiography performed during the action of these drugs demonstrated that the marginal sinus filled with contrast for a considerably longer time, sometimes up to 25 s. DISCUSSION MacNeal et al. (1927) studied the circulation of human and animal spleens using a technique of fixation of the spleen in a distended state and so were able to identify the circulatory pathways. These authors concluded that these vascular pathways enabled an intimate contact between the formed elements of the blood and the reticuloendothelial cells of the marginal zone. The anglographic studies reported here have demonstrated the importance of the marginal zone as a major blood pathway. Interest in the marginal zone has also been aroused in recent years following the demonstration of rapid localisation of injected antigens in the perifollicular region (Nossal et al., 1966; Mitchell and Abbott, 1971). Mitchell (1972) also demonstrated the migration of antigen-binding cells, prepared in vitro and injected in vivo, from the marginal zone to the germinal centre. Angiography has demonstrated that the marginal zones remain filled with contrast for a considerable period of time, haemodynamically. This delay in passage of the contrast media would provide a mechanism whereby there is prolonged contact of blood and particulate matter including antigen with the reticulo-endothelial cells of the marginal zone. Thus the perifollicular mononuclear phagocytes are
263
SPLEEN
-
•
a3
1_
M.s.
.......:.~ "~'~(,~.
':)":;":":'": :!':::
~
~ ~::
L.F. ".!....'
"V. V Fro. 9 An artist's impression of the splenic microvasculature, A, interlobular artery; al, a2 and a3, arterioles; MS, marginal sinus; CA, central arteriole; LF, lymphoid follicle; VS, venous sinus; V, vein. able to selectively assess particulate matter and cells in the blood for antigen processing or phagocytosis. MacNeal (1929) described two types of arteriole supplying the marginal sinus, namely, a branch of the interlobular artery arising before the lymphoid follicle and the terminations of the follicular capillaries. Our microfil studies have demonstrated a further arteriole supplying the marginal sinus which arises from the interlobular artery after it has passed through a lymphoid follicle. Whether these arteriolar branches of the interlobular arteries (al and as, Figs. 7, 9) correspond to the penicillar arteries described by many other authors is debatable. It has long been stated that the splenic arteries are end arteries and that there are no anastomoses between each lobule. Our angiographic studies have failed to show any anastomotic channels during normal flow or during physiological stimuli. However it has not been recognised previously that each follicular artery supplies a number of Malpighian bodies and this has been clearly shown by arteriography. MacNeal also suggested that these arterioles functioned intermittently similar to those of the skin (Krogh, 1921) and those of the renal capillary tufts (Richards, 1925). If perfusion of a single marginal zone was intermittent it would provide a mechanism whereby blood could be held in the sinus for a considerable period of time and thus explain the angiographic findings. The control of the mechanism has still to be elucidated.
264
CLINICAL RADIOLOGY
I f the b l o o d flow to the spleen is reduced by nerve stimulation at a high physiological frequency (5-10 Hz) or by intra-arterial injection of adrenaline or n o r a d r e n a l i n e (2-5-5 ~zg) the opacification o f the marginal zone at a n g i o g r a p h y can be disturbed. However, this m a y be due to the contraction of the smooth muscle in the trabeculae a n d capsule of the spleen rather t h a n alterations in the vascular pathway, because when similar vascular changes were obtained b y injecting drugs which do n o t have significant capsular activity such as Vasopressin a n d Angiotensin the m a r g i n a l sinuses are seen to fill clearly. I n the latter case the only change observed was that the sinuses r e m a i n e d full of contrast for a more prolonged period, sometimes u p to 25 s. Acknowledgements. - We are indebted to Mr W. Hinkes for his photographic skill, and to Miss F. Cuthill for the drawing. REFERENCES AYERS, A. B., DAVmS,B. N. & WITrmIN~TON,P. G. (1972). Responses of the isolated perfused human spleen to sympathetic nerve stimulation, catecholamines and polypeptides. British Journal o f Pharmacology, 44, 17-30. BJORKMAN,S. E. (1947). The splenic circulation with special reference to the function of the spleen sinus wall. Acta medica scandinavica, Supplementa 191. DAVIES, B. N., GAMBLE,J. & WIa~RIN~TON,P. G. (1968). Effects of noradrenaline, adrenaline and angiotensin on the vascular and capsular smooth muscle of the spleen of the dog. British Journal of Pharmacology and Chemotherapy, 32, 424P. DAWES, B. N., GAMBLE,J. & WITHRINGTON,P. G. (1973). Frequency-dependent differences in the responses of the capsular and vascular smooth muscle of the spleen of the dog to sympathetic nerve stimulation.Journal of Physiology (London), 228, 13-25. DAwEs, B. N. & WITHRIN~TON,P. G. (1968). The effects of
prostaglandin E1 and E2 on the smooth muscle of the dog spleen and on its response to catecholamines, angiotensin and nerve stimulation. British Journal of Pharmacology and Chemotherapy, 32, 136-144. KNISELY,M. H. (1936). Spleen studies: microscopic observation of the circulatory system of living unstimulated mammalian spleens. Anatomical Record, 65, 131-148. KROGn, A. (1921). Studies on the physiology of capillaries. II. The reactions to local stimuli of the blood vessels in the skin and web of the frog. Journal of Physiology (London), 55, 41~422. KRUMBrIAAR,E. B. (1926). Functions of the spleen. Physiological Reviews, VI, 160-200. LEwis, O. J. (1957). The blood vessels of the adult mammalian spleen. Journal of Anatomy, 91,245-250. MACNEAL,W. J. (1929). The circulation of blood through the spleen pulp. Archives of Pathology, 7, 215-227. MACNEAL, W. J., OTANI, S. & PATTERSON,M. B. (1927). The finer vascular channels of the spleen. American Journal of Pathology, 3, 111-112. McNEE J. W. (1931). The spleen: its structure function and diseases. Lancet, 220, 951-957, 1009-1015, 1063-1070. MITCHELL, J. (1972). Antigens in immunity. XVIII. The migration of antigen-binding, bone-marrow-derived and thymus derived spleen cells in mice. immunology, 22, 231-245. MITCHELL,J. & ABBOT,A. (1971). Antigens in immunity.XVI. A light and electron microscope study of antigen localisation in the rat spleen. Immunology, 21, 207-224. NISIMARU,Y. • STEGGARDA,F. R. (1932). Observations on the structure and function of certain blood vessels in the spleen. Journal of Physiology (London), 74, 327-337. NOSSAL, G. J. V., AUSTIN, C. M., PYE, J. & MITCHELL,J. (1966). Antigens in immunity. XII. Antigen trapping in the spleen. International Archives of Allergy and Applied Immunology, 29, 368-383. RICHARDS,A. N. (1925). The nature and mode of regulations of glomerular function. American Journal of Medical Science, 170, 781-803. ROBINSON, W. L. (1926). Vascular mechanism of the spleen. American Journal of Pathology, 2, 341-355. SNOOK, T. (1950). A comparative study of the vascular compartments in mammalian spleens. American Journal of Anatomy, 87, 31-77.