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An ultrastructural study of dopamine-immunoreactive nerve fibres in milky spots of the human greater omentum
ELSEVIER
Neuroscience Letters 168 (1994) 143-146
NEUROSCIHC[ LETTERS
An ultrastructural study of dopamine-immunoreactive nerve fibres in milky spots of the human greater omentum L.F.G. Krist a, I.L. Eestermans b, H.W.M. Steinbusch c, M A. Cuesta a, S. Meyer a'*, R.H.J. Beelen b ~Department of Surgery, Free University Hospital, P O. Box 7161, 1007 MC Amsterdam, The Netherlands bDepartment of Cell Biology, Faculty of Medicine, Free University, Amsterdam, The Netherlands ¢Department of Psychiatry and Neuropsychology, University,of Limburg, Maastricht, The Netherlands Received 25 June 1993; Revised version received 16 December 1993; Accepted 22 December 1993
Abstract
An ultrastructural study was performed to examine the presence and distribution of dopamine-immunoreactive nerve fibres in milky spots of the human greater omentum. Non-myelinated nerve fibres were located perivascularly as well as throughout the milky spots. Dopamine immunoreactivity was demonstrated in the nerve fibres and in a portion of the macrophage population. These results demonstrate a discrepancy between human and non-human milky spots. Key words: Immunocytochemistry; Innervation; Dopamine; Electron microscopy
The catecholamine dopamine (DA) is widely distributed in the central and peripheral nervous system. DA is a precursor of the neurotransmitter noradrenaline as well as a neurotransmitter itself. Moreover, D A has a regulatory effect on the size of blood vessels [10] and influences the immune system [4,5,7]. In homogenates of the greater omentum of dogs, high concentrations of D A have been demonstrated [8], Furthermore, a network of fine noradrenergic nerve fibres, surrounding arteries and veins, has been demonstrated in the human greater omentum [6]. Currently, no other data are available for the localization of monoaminergic nerve fibres in the greater omentum. Milky spots are present within the greater omentum. In animal studies it has been demonstrated that milky spots have characteristics of lymphoid tissue [2] and are comprised of macrophages, lymphocytes and granulocytes [1,3]. Little is known about the functional and structural characteristics of milky spots in the human greater omentum. A detailed description of the distribution of DA-containing nerve fibres in milky spots has not yet been re-
* Corresponding author. Fax: (31) (20) 548 4076. 0304-3940194l$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0 3 0 4 - 3 9 4 0 ( 9 3 ~E0894-2
ported. The present study examines the presence and ultrastructural distribution of DA-immunoreactive nerve fibres in milky spots of the human greater omentum. Specimens of transparent parts of human greater omentum were taken from three patients (age 1, 6 and 28 years; 2 males and 1 female) suffering from reflux oesophagitis (2) and pancreatic carcinoma (1). In all cases, the greater omentum was unaffected by the disease of the patient. Furthermore, patients did not receive any DA-related drugs. To examine the morphology of milky spots, the omentum of one patient was rinsed in phosphate-buffered saline (PBS; pH 7.4) and fixed in 1.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 8 h. After fixation, the omentum was examined in a PBS buffer and milky spots were isolated, postfixed in 1% OsO4, dehydrated, impregnated and finally embedded in epoxy resin according to standard procedures. Ultrathin sections (700/k) were cut on an ultramicrotome (Reicheft) and stained with uranyl acetate and lead citrate. All specimens were examined in a T E M 301 (Philips) at 60 kV. To demonstrate D A in the nerve fibres of milky spots omental biopsies of two patients were processed on separate occasions. The biopsies were fixed directly in 5%
~i~ • ~ ~
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~ ~i~~
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L.EG. Krist et al. INeuroscience Letters 168 (1994) 143 146
glutaraldehyde in 0.1% sodium cacodylate buffer with 1% Na2S205 (pH 7.4) on ice, for 30 min [12]. Milky spots were isolated and these specimens were rinsed in 0.1 M Tris-buffered saline (TBS; pH 7.6) with 1% Na2S205 and were incubated for 16 h at 4°C with a rabbit antibody against DA, diluted 1:3000 [12]. After rinsing with TBS, specimens were incubated with biotinylated horse antirabbit antibody (Vectastain, ABC kit, Vector Laboratories, USA). To prevent endogenous peroxidase staining, specimens were preincubated with 0.3% H202 in methanol. Incubation with avidin-biotin-peroxidase complex (Vectastain, ABC kit) took place for 45 min at room temperature. The trapping agent for developing a precipitation product consisted of diaminobenzidine 0.5 mg/ml in 0.1 M PBS + 0.03% H202. Between previous steps, specimens were rinsed with 0.1 M Tris-HC1. After storage overnight in 5% glutaraldehyde, the tissue was postfixed in OsO 4 for 1 h at 4°C. Dehydration, impregnation and embedding in epoxy resin were performed as described above. As a negative control, the first antibody was omitted in each series. Specimens were not counterstained. Non-myelinated nerve fibres which contained vesicles of different sizes were located perivascularly as well as throughout the milky spots. The nerve fibres were in close apposition with the cells present in the milky spots (Fig. 1). DA-immunoreactive nerve fibres contained both mildly and strongly stained vesicles of varying diameter (0.07-0.10 pm) and a diffuse shadowing of reaction product associated with membrane like structures (Fig. 2). Some macrophages were stained as well: in +40% of this cell type, reaction product could clearly be seen inside endosomes. These macrophages were located mainly in the presence of capillaries (Fig. 3). There was no difference in the density or distribution of the DA-containing nerve fibres and macrophages in omentum obtained from patients with reflux oesophagites and carcinoma of the pancreas: both separately processed specimens showed the same staining pattern. The present findings demonstrate for the first time the innervation of milky spots in the human greater omenturn. This is of considerable significance in view of the fact that extensive investigations of milky spots in the greater omentum of animals never revealed any nerve tissue [1,3,9], which indicates a structural difference be-
145
tween human and non-human milky spots in the greater omentum. DA immunoreactivity was demonstrated in nerve fibres and in some macrophages. Although DA is an easily diffusable molecule, the applied fixation technique secured localization of DA at the time the omentum biopsy was taken [12]. Furthermore, the applied antibody against DA has a high specificity and does not show cross-reactivity towards other monoamines including noradrenaline [12]. Although DA is a precursor of NA, it is unlikely that we demonstrated DA in noradrenergic fibres for two reasons: (A) the enzymatic conversion of dopamine to noradrenaline is not at equilibrium, in other words, as soon as dopamine has been synthesised from tyrosine the conversion to NA proceeds. The rate limiting enzyme in this pathway is tyrosine-hydroxylase. (B) In rat studies using the same antibody no dopamine immunoreactivity could be detected in noradrenerglc brain structures [3]. The functional significance of DA-containing nerve fibres and macrophages within the milky spots is unknown. Okamura et al. [10] demonstrated that isolated human epiploic veins respond to DA only with contraction, while human epiploic arteries in the same region of the omentum respond with relaxation. This will influence the blood flow in the omentum and will have a profound effect on the number of leucocytes reaching the inside of milky spots via the blood stream [11]. The presence of perivascularly located DA-containing nerve fibres in milky spots might indicate a local regulation of homing of leucocytes. The presence of DA immunoreactivity within some of the macrophage population in milky spots might indicate a role of DA as an immune system effector [4,5,7]. We thank Mr. Shimon Paniry for expert photographical assistance, Mr. James Andrick for correcting the manuscript and Dr. J.M.L. Henselmans for her advice.
[1] Beelen, R.H.J., Fluitsma, D.M. and Hoefsmit, E.C.M., Cellular composition of milky spots and the ultrastructure of milky spots macrophages and reticulum cells, J. Reticuloendothel. Soc., 28 (1980) 585-599. [2] Beelen, R.H.J., Hendrickx, R.J.B.M., Eestermans, I.L. and Wijffels, J.F.A.M., Milky spots can be considered as omentum associated lymphoid tissue. In B. Imhof (Ed.t, Lymphatic Tissues
(.._ Fig. 1. Electron micrograph of a nerve fibre stained with uranyl acetate and lead citrate situated adjacent to a macrophage (M¢). Within the nerve fibre mitochondria (m), clear vesicles (cv) and granulated vesicles (gv) are present. Fig. 2. Dopaminergic nerve fibre with precipitation product of the DA antibody located in vesicles (v) and in association with me~abrane-like structures (arrow). No counterstaining has been performed. Fig. 3. Macrophage (Mr,0) with precipitation product of the DA antibody, located in a variable degree in endosomes (el. This cell is in close apposition with an endothelial cell (E). No counterstaining has been performed. Bars = 0.2/lm.
146
[3] [4] [5]
[6]
[7]
[8]
L E G . Krist et al. I Neuroxciem'~ Letters 168 (1994) 143 146
and In-Vivo Immune Responses, Dekker, New York, 1991, pp. 519-524. Carr, I., The fine structure of the ceils of the mouse peritoneum, Z. ZeUforsch,, 80 (1967) 534-555. Cotzias, G.C., Miller, S.T., Tang, L.C. and Papavasiliou. P.S., Levadopa, fertility and longevity, Science, 196 (1977) 549-551. Cotzias, G.C, and Tang, L.C., An adenylate cyclase of brain reflects propensity for breast cancer in mice, Science, 197 (19771 1094-1096, Edvinsson, L., Hakanson, R., Steen, S., Sundler, F., Uddman, R. and Wahlestedt, C., Innervation of human omental arteries and veins and vasomotor responses to noradrenaline, neuropeptide Y, substance P and vasoactive intestinal peptide, Regnl. Pept., 12 (1985) 67-79. Felten, D.L., Neurotransmitter signalling of cells of the immune system: important progress, major gaps, Brain Behav., 5 (1991) 2-8. Goldsmith, H.S., Mclntosh, T., Vezina, R.M. and Colton, Th., Vasoactive neurochemicals identified in omentum: a preliminary report, Br. J. Neurosurg., 1 (1987) 359 364.
[9] Lang, J., Uber die Gefasse und die Zellen der Milchflecken, Z. Zellforsch.. 66 (1965) 1-27. [10] Okamura, T., Yamazaki, M. and Toda, N., Response to dopamine of isolated human and monkey veins compared with those of the arteries, J. Pharmacol. Exp. Ther., 258 (1991) 275 -279. [11] Ottaway, C.A., Neurophysiological events and lymphocyte migration and distribution in vivo. In E.T. Goetzl and N.H. Spector (Eds.), Neuroimmune Networks: Physiology and Disease, Alan R Liss, New York, 1989, pp. 235-241. [12] Steinbusch, H.W.M., Van Vliet, S.P., Bol, J.G.J.M. and De Vente, J., Development and application of antibodies to primary (DA, I~-DOPA) and secondary (cGMP) messengers: a technical report. In A. Calas and D. Eugene (Eds.), Neurocytochemicat Methods, Springer, Berlin, 1991, pp. 1 27. [13] Steinbusch, H.W.M., Wouterlood F.G., De Vente, J., Bol, J.G.J.M. and Berkenbosch, F. and Van Vliet, S.P., Immunohistochemical localization of monoamines and cyclic nucleotides. Their application in quantitative immunofluorescence studies and tracing monoaminergic neuronal connections. Acta Histochem.. Suppl. 35 (1988) 85-106.
Neuroscience Letters 168 (1994) 143-146
NEUROSCIHC[ LETTERS
An ultrastructural study of dopamine-immunoreactive nerve fibres in milky spots of the human greater omentum L.F.G. Krist a, I.L. Eestermans b, H.W.M. Steinbusch c, M A. Cuesta a, S. Meyer a'*, R.H.J. Beelen b ~Department of Surgery, Free University Hospital, P O. Box 7161, 1007 MC Amsterdam, The Netherlands bDepartment of Cell Biology, Faculty of Medicine, Free University, Amsterdam, The Netherlands ¢Department of Psychiatry and Neuropsychology, University,of Limburg, Maastricht, The Netherlands Received 25 June 1993; Revised version received 16 December 1993; Accepted 22 December 1993
Abstract
An ultrastructural study was performed to examine the presence and distribution of dopamine-immunoreactive nerve fibres in milky spots of the human greater omentum. Non-myelinated nerve fibres were located perivascularly as well as throughout the milky spots. Dopamine immunoreactivity was demonstrated in the nerve fibres and in a portion of the macrophage population. These results demonstrate a discrepancy between human and non-human milky spots. Key words: Immunocytochemistry; Innervation; Dopamine; Electron microscopy
The catecholamine dopamine (DA) is widely distributed in the central and peripheral nervous system. DA is a precursor of the neurotransmitter noradrenaline as well as a neurotransmitter itself. Moreover, D A has a regulatory effect on the size of blood vessels [10] and influences the immune system [4,5,7]. In homogenates of the greater omentum of dogs, high concentrations of D A have been demonstrated [8], Furthermore, a network of fine noradrenergic nerve fibres, surrounding arteries and veins, has been demonstrated in the human greater omentum [6]. Currently, no other data are available for the localization of monoaminergic nerve fibres in the greater omentum. Milky spots are present within the greater omentum. In animal studies it has been demonstrated that milky spots have characteristics of lymphoid tissue [2] and are comprised of macrophages, lymphocytes and granulocytes [1,3]. Little is known about the functional and structural characteristics of milky spots in the human greater omentum. A detailed description of the distribution of DA-containing nerve fibres in milky spots has not yet been re-
* Corresponding author. Fax: (31) (20) 548 4076. 0304-3940194l$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0 3 0 4 - 3 9 4 0 ( 9 3 ~E0894-2
ported. The present study examines the presence and ultrastructural distribution of DA-immunoreactive nerve fibres in milky spots of the human greater omentum. Specimens of transparent parts of human greater omentum were taken from three patients (age 1, 6 and 28 years; 2 males and 1 female) suffering from reflux oesophagitis (2) and pancreatic carcinoma (1). In all cases, the greater omentum was unaffected by the disease of the patient. Furthermore, patients did not receive any DA-related drugs. To examine the morphology of milky spots, the omentum of one patient was rinsed in phosphate-buffered saline (PBS; pH 7.4) and fixed in 1.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 8 h. After fixation, the omentum was examined in a PBS buffer and milky spots were isolated, postfixed in 1% OsO4, dehydrated, impregnated and finally embedded in epoxy resin according to standard procedures. Ultrathin sections (700/k) were cut on an ultramicrotome (Reicheft) and stained with uranyl acetate and lead citrate. All specimens were examined in a T E M 301 (Philips) at 60 kV. To demonstrate D A in the nerve fibres of milky spots omental biopsies of two patients were processed on separate occasions. The biopsies were fixed directly in 5%
~i~ • ~ ~
i4!I ~~ -~
~ ~i~~
~ L~
L.EG. Krist et al. INeuroscience Letters 168 (1994) 143 146
glutaraldehyde in 0.1% sodium cacodylate buffer with 1% Na2S205 (pH 7.4) on ice, for 30 min [12]. Milky spots were isolated and these specimens were rinsed in 0.1 M Tris-buffered saline (TBS; pH 7.6) with 1% Na2S205 and were incubated for 16 h at 4°C with a rabbit antibody against DA, diluted 1:3000 [12]. After rinsing with TBS, specimens were incubated with biotinylated horse antirabbit antibody (Vectastain, ABC kit, Vector Laboratories, USA). To prevent endogenous peroxidase staining, specimens were preincubated with 0.3% H202 in methanol. Incubation with avidin-biotin-peroxidase complex (Vectastain, ABC kit) took place for 45 min at room temperature. The trapping agent for developing a precipitation product consisted of diaminobenzidine 0.5 mg/ml in 0.1 M PBS + 0.03% H202. Between previous steps, specimens were rinsed with 0.1 M Tris-HC1. After storage overnight in 5% glutaraldehyde, the tissue was postfixed in OsO 4 for 1 h at 4°C. Dehydration, impregnation and embedding in epoxy resin were performed as described above. As a negative control, the first antibody was omitted in each series. Specimens were not counterstained. Non-myelinated nerve fibres which contained vesicles of different sizes were located perivascularly as well as throughout the milky spots. The nerve fibres were in close apposition with the cells present in the milky spots (Fig. 1). DA-immunoreactive nerve fibres contained both mildly and strongly stained vesicles of varying diameter (0.07-0.10 pm) and a diffuse shadowing of reaction product associated with membrane like structures (Fig. 2). Some macrophages were stained as well: in +40% of this cell type, reaction product could clearly be seen inside endosomes. These macrophages were located mainly in the presence of capillaries (Fig. 3). There was no difference in the density or distribution of the DA-containing nerve fibres and macrophages in omentum obtained from patients with reflux oesophagites and carcinoma of the pancreas: both separately processed specimens showed the same staining pattern. The present findings demonstrate for the first time the innervation of milky spots in the human greater omenturn. This is of considerable significance in view of the fact that extensive investigations of milky spots in the greater omentum of animals never revealed any nerve tissue [1,3,9], which indicates a structural difference be-
145
tween human and non-human milky spots in the greater omentum. DA immunoreactivity was demonstrated in nerve fibres and in some macrophages. Although DA is an easily diffusable molecule, the applied fixation technique secured localization of DA at the time the omentum biopsy was taken [12]. Furthermore, the applied antibody against DA has a high specificity and does not show cross-reactivity towards other monoamines including noradrenaline [12]. Although DA is a precursor of NA, it is unlikely that we demonstrated DA in noradrenergic fibres for two reasons: (A) the enzymatic conversion of dopamine to noradrenaline is not at equilibrium, in other words, as soon as dopamine has been synthesised from tyrosine the conversion to NA proceeds. The rate limiting enzyme in this pathway is tyrosine-hydroxylase. (B) In rat studies using the same antibody no dopamine immunoreactivity could be detected in noradrenerglc brain structures [3]. The functional significance of DA-containing nerve fibres and macrophages within the milky spots is unknown. Okamura et al. [10] demonstrated that isolated human epiploic veins respond to DA only with contraction, while human epiploic arteries in the same region of the omentum respond with relaxation. This will influence the blood flow in the omentum and will have a profound effect on the number of leucocytes reaching the inside of milky spots via the blood stream [11]. The presence of perivascularly located DA-containing nerve fibres in milky spots might indicate a local regulation of homing of leucocytes. The presence of DA immunoreactivity within some of the macrophage population in milky spots might indicate a role of DA as an immune system effector [4,5,7]. We thank Mr. Shimon Paniry for expert photographical assistance, Mr. James Andrick for correcting the manuscript and Dr. J.M.L. Henselmans for her advice.
[1] Beelen, R.H.J., Fluitsma, D.M. and Hoefsmit, E.C.M., Cellular composition of milky spots and the ultrastructure of milky spots macrophages and reticulum cells, J. Reticuloendothel. Soc., 28 (1980) 585-599. [2] Beelen, R.H.J., Hendrickx, R.J.B.M., Eestermans, I.L. and Wijffels, J.F.A.M., Milky spots can be considered as omentum associated lymphoid tissue. In B. Imhof (Ed.t, Lymphatic Tissues
(.._ Fig. 1. Electron micrograph of a nerve fibre stained with uranyl acetate and lead citrate situated adjacent to a macrophage (M¢). Within the nerve fibre mitochondria (m), clear vesicles (cv) and granulated vesicles (gv) are present. Fig. 2. Dopaminergic nerve fibre with precipitation product of the DA antibody located in vesicles (v) and in association with me~abrane-like structures (arrow). No counterstaining has been performed. Fig. 3. Macrophage (Mr,0) with precipitation product of the DA antibody, located in a variable degree in endosomes (el. This cell is in close apposition with an endothelial cell (E). No counterstaining has been performed. Bars = 0.2/lm.
146
[3] [4] [5]
[6]
[7]
[8]
L E G . Krist et al. I Neuroxciem'~ Letters 168 (1994) 143 146
and In-Vivo Immune Responses, Dekker, New York, 1991, pp. 519-524. Carr, I., The fine structure of the ceils of the mouse peritoneum, Z. ZeUforsch,, 80 (1967) 534-555. Cotzias, G.C., Miller, S.T., Tang, L.C. and Papavasiliou. P.S., Levadopa, fertility and longevity, Science, 196 (1977) 549-551. Cotzias, G.C, and Tang, L.C., An adenylate cyclase of brain reflects propensity for breast cancer in mice, Science, 197 (19771 1094-1096, Edvinsson, L., Hakanson, R., Steen, S., Sundler, F., Uddman, R. and Wahlestedt, C., Innervation of human omental arteries and veins and vasomotor responses to noradrenaline, neuropeptide Y, substance P and vasoactive intestinal peptide, Regnl. Pept., 12 (1985) 67-79. Felten, D.L., Neurotransmitter signalling of cells of the immune system: important progress, major gaps, Brain Behav., 5 (1991) 2-8. Goldsmith, H.S., Mclntosh, T., Vezina, R.M. and Colton, Th., Vasoactive neurochemicals identified in omentum: a preliminary report, Br. J. Neurosurg., 1 (1987) 359 364.
[9] Lang, J., Uber die Gefasse und die Zellen der Milchflecken, Z. Zellforsch.. 66 (1965) 1-27. [10] Okamura, T., Yamazaki, M. and Toda, N., Response to dopamine of isolated human and monkey veins compared with those of the arteries, J. Pharmacol. Exp. Ther., 258 (1991) 275 -279. [11] Ottaway, C.A., Neurophysiological events and lymphocyte migration and distribution in vivo. In E.T. Goetzl and N.H. Spector (Eds.), Neuroimmune Networks: Physiology and Disease, Alan R Liss, New York, 1989, pp. 235-241. [12] Steinbusch, H.W.M., Van Vliet, S.P., Bol, J.G.J.M. and De Vente, J., Development and application of antibodies to primary (DA, I~-DOPA) and secondary (cGMP) messengers: a technical report. In A. Calas and D. Eugene (Eds.), Neurocytochemicat Methods, Springer, Berlin, 1991, pp. 1 27. [13] Steinbusch, H.W.M., Wouterlood F.G., De Vente, J., Bol, J.G.J.M. and Berkenbosch, F. and Van Vliet, S.P., Immunohistochemical localization of monoamines and cyclic nucleotides. Their application in quantitative immunofluorescence studies and tracing monoaminergic neuronal connections. Acta Histochem.. Suppl. 35 (1988) 85-106.