1290
found that viable adherent splenic macrophages from oil-treated BALB/c mice could produce a factor which, in
with
cells in epithelial tumours commonly occurs in organs with large reticuloendothelial beds of macrophages-i.e., lung and liver. Also, human malignant effusions contain numerous macrophages, which we have found capable of stimulating tumour-colony
2-mercaptoethanol (2-ME), promoted soft-agar colony formation by human myeloma cells.8 Immunological studies established that the conditioning factor was not antigenically related to the colony-stimulating factor needed for normal granulocyte colony formation.8 During studies of the growth specificity of the conditioning factor, we were surprised to find that medium conditioned by the oil-primed BALB/c macrophages supported in-vitro tumour-colony growth from fresh biopsy specimens of a wide variety of cancers.9 These included myeloma, lymphoma, melanoma, and
growth. Our hypothesis should be easily testable by a variety of biological experiments both in vitro and in vivo. Furthermore, isolation and definition of the putative macrophage-derived clonal-proliferation factor or factors could provide a new understanding of the clonal expansion of immunocytes as well as of the proliferation and metastasis of neoplastic cells.
neuroblastoma, as well as carcinomas of the ovary, endometrium, lung, and other sites.9 We also found that
CA-14102, CA-17094, and CA-21839 from the National Cancer Insti-
concert
depletion of the macrophage population (by phagocytosis of carbonyl iron) from malignant ascites in ovarian carcinoma greatly reduced ovarian-tumour colony growth.1O This provided evidence that in these cases the patients’ autologous macrophages also enhanced tumour-colony growth. Our in-vitro work on the role of the macrophage in promoting tumour-colony formation has had a remarkable parallel with studies of normal murine B-lymphocyte colony formation," which also requires 2-mercaptoethanol 12 and is promoted by a soluble factor’3 elaborated by macrophages. In addition, secretory products from macrophages can promote the proliferation of antibody-forming cells. 14 The fact that macrophages promote both normal B-lymphocyte and tumour colony formation via a soluble factor has led us to propose the following hypothesis schematically shown in the accompanying figure. We propose that both immune and tumour clones require a common and specific growth stimulus which promotes clonal proliferation of an already-triggered hypermutable cell population. The evolutionary devela growth promoter by the macrophage presumably have positive survival advantage by allowing amplification of the appropriate clones of immune cells in response to foreign antigens. This factor would also incidentally stimulate the growth of clones of tumour cells that have been previously oncogenically
opment of such
would
transformed. It is therefore of interest that an elaborate adaptive immune mechanism and diverse cancers appear to have evolved at similar points in the vertebrate phy-
logeny.15 Some clinical features of metastasis may also be consistent with the role of the macrophage as a promoter of tumour growth. For example, metastasis of clonogenic
The
macrophage
as a
two-edged
sword in immnnity and
cancer.
A proliferation factor elaborated to support clonal multiplication of antigen-triggered B-lymphocytes also incidentally supports proliferation of neoplastic cells. Membrane proteins from dying cancer cells further stimulate elaboration of this factor by macrophages.
This work is
supported by
Public Health Service grants
nos.
tute, U.S.A.
Requests for reprints should be addressed to S.E.S., Section of and Oncology, Department of Internal Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724,
Hematology U.S.A.
REFERENCES 1. Boutwell, R. K. C.R.C. crit. Rev. Toxicol. 1974, 2, 419. 2. Clark-Lewis, I., Murray, A. Cancer Res. 1978, 38, 494. 3. Potter, M. Semin. Hemat. 1973, 10, 19. 4. Cancro, M., Potter, M. J. exp. Med. 1970, 131, 429 5. Namba, Y., Hanaoka, M. J. Immun. 1972, 109, 1193. 6. Namba, Y., Hanaoka, M. Cell. Immun. 1974, 12, 74. 7. Potter, M. Physiol. Rev. 1972, 52, 631. 8. Hamburger, A., Salmon, S. E. J. clin. Invest. 1977, 60, 846. 9. Hamburger, A. W., Salmon, S. E. Science, 1977, 197, 461. 10. Salmon, S. E., Hamburger, A. W., Soehnlen, B., Schmidt, J., Alberts, D. S. Proc. Am. Ass. Cancer Res. 1978, 19, 920. 11. Metcalf, D., Warner, N. L., Nossal, G. J. V., Miller, J. F. A. P., Shortman, K., Rabellino, E. Nature, 1975, 255, 630. 12. Metcalf, D. J. Immun. 1976, 166, 635. 13. Kurland, J. E., Kincade, P. W., Moore, M. A. S. J. exp. Med. 1977, 146,
1420. 14. Unanuae, E. Am. J. Path. 1976, 83, 396. 15. Balls, M., Ruben, L. in Comparative lonis); p. 167. New York, 1976.
Immunology (edited by J.
Marcha-
Methods and Devices ASSESSMENT OF PELVIC-FLOOR DISORDERS AND INCONTINENCE BY ELECTROPHYSIOLOGICAL RECORDING OF THE ANAL REFLEX MICHAEL M. HENRY MICHAEL SWASH
Departments of Surgery and Neurology, Hospital, London EC1V 2PS,
St. Mark’s
and The London
Hospital,
London E1 1BB
THE anal reflex was first described by Rossolimol at the end of the last century but has attracted little attention since then, although its absence is a well-known sign of lesions in the cauda equina or sacral spinal cord.2 Histological studies indicate that denervation of the external anal sphincter and puborectalis muscles occurs in many patients with idiopathic faecal incontinence.3 It has been suggested that this might result from entrapment and stretch-induced injury to the pudendal nerves.3,4 To test this suggestion we recorded the motor-unit action potential associated with the anal reflex, since damaged pudendal nerves might show abnormalities of latency, amplitude, or duration of the reflex response. We describe here a simple electrophysiological method for recording the anal reflex in man, and present preliminary data on its latency in 13 normal subjects (aged 22-75 yr, mean 43), 10 of whom were female, and in patients with various disorders affecting the pelvic-floor musculature. Since such disorders are commoner in women than in men, we have deliberately studied more women than men. Methods The subjects were placed in the left lateral position. A con-
1291 centric needle E.M.G. electrode was inserted into the superficial portion of the external anal sphincter muscle in the midline
position posterior to the anal verge, and the perianal skin was stimulated by a bipolar surface electrode (standard ’DISA 14A21’ E.M.G. apparatus). A ground electrode was applied to the thigh. The reflexly evoked muscle action potentials were displayed on the oscilloscope of the E.M.G. apparatus or on a separate ’Telequipment DM 64’ storage oscilloscope. Squarewave stimuli of 0-1 1 to 0-2 ms duration and of variable voltage were used. The reflexly evoked electrical responses detected by the needle electrode in the external anal sphincter muscle were generally maximum with stimuli of about 90 V.
Results’ The muscle action potential in a normal anal reflex recorded the oscilloscope (fig. 1) coincided with contraction of the external anal sphincter muscle, recognised by transient constriction of the anal margin. The latency of the electrical response in this record was 7.9 ms. The ranges and mean values of the latencies of the anal reflex in normal subjects are as follows : on
There
changes in of the responses
were no
amplitudes
latency ranged
with increasing age. The from 50 to 500 fLV (mean
125V). The reflex response to a single stimulus is usually readily recognisable (fig. 1), but the random basal activity in the normal external anal sphincter5 may sometimes make identification of the reflexly evoked muscle action potential difficult. In some patients the reflex response is of such low amplitude that it approximates to that of basal contractions. Repeated supramaximum stimuli (usually about 90 V), delivered at about 1/s, can then be used to evoke a reflex response with a conspicuous,
latency, which can be readily isolated from basal contractions of the sphincter muscle (fig. 2). Both the interval between stimulus and response and the form of the response suggest that the latter is not an artefact resulting from direct spread of current from the stimulating to the recording electrode. However, since the two electrodes were only about 6 cm apart, we tested this possibility. In one patient, therefore, we infiltrated the skin underlying the stimulating electrode with 2 ml of 1% xylocaine. This produced an area of complete anaesthesia under the stimulating electrode at the anal margin and abolished the reflex response for about 30 min. The basal potentials, which were of much smaller amplitude than the reflex itself, were not affected by the local anoesthesia. A similar injection of normal saline, at a later date, did not alter the reflex response. The latency of the response was constant when supramaximum stimuli were used (fig. 2), and this was also the shortest latency recorded in each subject. Stimuli of lesser amplitude produced longer latencies. That the latency cannot be reduced further was confirmed in 1 subject by measuring it immediately after a supramaximum tetanus had been applied to the stimulation point on the perianal skin. No change in latency resulted after this procedure.
constant
Discussion This
simple electrophysiological method for studying the of the anal reflex, and the amplitude and duration of the response, gives reproducible results in individual subjects. It is not uncomfortable if the perianal cutaneous stimuli are applied less frequently than about 1/s. The afferent pathways of the anal reflex lie in the perianal branch of the pudendal nerves, which synapse in S2 and S3 segments of the spinal cord,’’6’’and the efferent pathway in the pudendal nerves and their perineal and inferior rectal branches.4,6 The response is detected in the external anal sphincter muscles and probably also the puborectalis and part of the levator ani muscles.6 Since the anal reflex, like the abdominal reflex,8 is evoked by cutaneous stimulation, it is probably polysynaptic. The period of central synaptic delay in polysynaptic reflexes is variable. When recording the latency of the anal reflex it is important, therefore, to establish the shortest possible latency which can be done by ensuring that the cutaneous stimulus is supramaximum, since prior tetanisation of the perianal skin did not shorten the latency. The reflex mechanisms underlying faecal continence are of fundamental importance but they have been little studied since the anorectal balloon pressure recordings of Denny-Brown and Robertson.9 A recording of the anal reflex in a patient with idiopathic faecal incontinence shows that the latency of the reflex was substantially increased (20 ms) and its amplitude (20 fL V) was less than that of normal subjects (fig. 2). These observations suggest that electrophysiological recording of the anal reflex in man may prove useful in assessing the function of the nerves supplying the muscles of the pelvic floor, especially in patients with fsecal incontinence and with descending perineum syndrome in whom stretch-induced injury to the pudendal nerves, perhaps associated with entrapment,3 might lead to detectable changes in latency, amplitude, or duration of the evoked response.
latency
We thank Sir Alan Parks for his encouragement. M. M. H. is sup-
ported by the St. Mark’s Hospital Research Foundation. REFERENCES
1. Aminev, A. M., Amineva, V. A. J. Procrol. 1969, 20, 339. 2. Walton, J. N. in Brain’s Diseases of the Nervous System;
Fig. 2-Anorectal incontinence. Arrow indicates anal reflex
response at
a constant
1/s). Other complexes are "basal" potentials Latency in this case is 19.2ms.
latency (stimuli sphincter.
of anal
1977. 3. Parks, A. G., Swash, M., Urich, H. Gut, 1977, 18, 656. 4. Beersiek, F., Swash, M. Unpublished. 5. Floyd, W. F., Walls, E. W. J. Physiol. 1953, 122, 599. 6. Bishop, B. Neurophysiology, 1959, 22, 679. 7. Sherrington, C. S. J. Physiol. 1892, 13, 672. 8. Kugelberg, E., Hagbarth, K. E. Brain, 1958, 81, 290. 9. Denny-Brown, D., Robertson, G. ibid, 1935, 58, 256.
p. 751. Oxford.
found that viable adherent splenic macrophages from oil-treated BALB/c mice could produce a factor which, in
with
cells in epithelial tumours commonly occurs in organs with large reticuloendothelial beds of macrophages-i.e., lung and liver. Also, human malignant effusions contain numerous macrophages, which we have found capable of stimulating tumour-colony
2-mercaptoethanol (2-ME), promoted soft-agar colony formation by human myeloma cells.8 Immunological studies established that the conditioning factor was not antigenically related to the colony-stimulating factor needed for normal granulocyte colony formation.8 During studies of the growth specificity of the conditioning factor, we were surprised to find that medium conditioned by the oil-primed BALB/c macrophages supported in-vitro tumour-colony growth from fresh biopsy specimens of a wide variety of cancers.9 These included myeloma, lymphoma, melanoma, and
growth. Our hypothesis should be easily testable by a variety of biological experiments both in vitro and in vivo. Furthermore, isolation and definition of the putative macrophage-derived clonal-proliferation factor or factors could provide a new understanding of the clonal expansion of immunocytes as well as of the proliferation and metastasis of neoplastic cells.
neuroblastoma, as well as carcinomas of the ovary, endometrium, lung, and other sites.9 We also found that
CA-14102, CA-17094, and CA-21839 from the National Cancer Insti-
concert
depletion of the macrophage population (by phagocytosis of carbonyl iron) from malignant ascites in ovarian carcinoma greatly reduced ovarian-tumour colony growth.1O This provided evidence that in these cases the patients’ autologous macrophages also enhanced tumour-colony growth. Our in-vitro work on the role of the macrophage in promoting tumour-colony formation has had a remarkable parallel with studies of normal murine B-lymphocyte colony formation," which also requires 2-mercaptoethanol 12 and is promoted by a soluble factor’3 elaborated by macrophages. In addition, secretory products from macrophages can promote the proliferation of antibody-forming cells. 14 The fact that macrophages promote both normal B-lymphocyte and tumour colony formation via a soluble factor has led us to propose the following hypothesis schematically shown in the accompanying figure. We propose that both immune and tumour clones require a common and specific growth stimulus which promotes clonal proliferation of an already-triggered hypermutable cell population. The evolutionary devela growth promoter by the macrophage presumably have positive survival advantage by allowing amplification of the appropriate clones of immune cells in response to foreign antigens. This factor would also incidentally stimulate the growth of clones of tumour cells that have been previously oncogenically
opment of such
would
transformed. It is therefore of interest that an elaborate adaptive immune mechanism and diverse cancers appear to have evolved at similar points in the vertebrate phy-
logeny.15 Some clinical features of metastasis may also be consistent with the role of the macrophage as a promoter of tumour growth. For example, metastasis of clonogenic
The
macrophage
as a
two-edged
sword in immnnity and
cancer.
A proliferation factor elaborated to support clonal multiplication of antigen-triggered B-lymphocytes also incidentally supports proliferation of neoplastic cells. Membrane proteins from dying cancer cells further stimulate elaboration of this factor by macrophages.
This work is
supported by
Public Health Service grants
nos.
tute, U.S.A.
Requests for reprints should be addressed to S.E.S., Section of and Oncology, Department of Internal Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724,
Hematology U.S.A.
REFERENCES 1. Boutwell, R. K. C.R.C. crit. Rev. Toxicol. 1974, 2, 419. 2. Clark-Lewis, I., Murray, A. Cancer Res. 1978, 38, 494. 3. Potter, M. Semin. Hemat. 1973, 10, 19. 4. Cancro, M., Potter, M. J. exp. Med. 1970, 131, 429 5. Namba, Y., Hanaoka, M. J. Immun. 1972, 109, 1193. 6. Namba, Y., Hanaoka, M. Cell. Immun. 1974, 12, 74. 7. Potter, M. Physiol. Rev. 1972, 52, 631. 8. Hamburger, A., Salmon, S. E. J. clin. Invest. 1977, 60, 846. 9. Hamburger, A. W., Salmon, S. E. Science, 1977, 197, 461. 10. Salmon, S. E., Hamburger, A. W., Soehnlen, B., Schmidt, J., Alberts, D. S. Proc. Am. Ass. Cancer Res. 1978, 19, 920. 11. Metcalf, D., Warner, N. L., Nossal, G. J. V., Miller, J. F. A. P., Shortman, K., Rabellino, E. Nature, 1975, 255, 630. 12. Metcalf, D. J. Immun. 1976, 166, 635. 13. Kurland, J. E., Kincade, P. W., Moore, M. A. S. J. exp. Med. 1977, 146,
1420. 14. Unanuae, E. Am. J. Path. 1976, 83, 396. 15. Balls, M., Ruben, L. in Comparative lonis); p. 167. New York, 1976.
Immunology (edited by J.
Marcha-
Methods and Devices ASSESSMENT OF PELVIC-FLOOR DISORDERS AND INCONTINENCE BY ELECTROPHYSIOLOGICAL RECORDING OF THE ANAL REFLEX MICHAEL M. HENRY MICHAEL SWASH
Departments of Surgery and Neurology, Hospital, London EC1V 2PS,
St. Mark’s
and The London
Hospital,
London E1 1BB
THE anal reflex was first described by Rossolimol at the end of the last century but has attracted little attention since then, although its absence is a well-known sign of lesions in the cauda equina or sacral spinal cord.2 Histological studies indicate that denervation of the external anal sphincter and puborectalis muscles occurs in many patients with idiopathic faecal incontinence.3 It has been suggested that this might result from entrapment and stretch-induced injury to the pudendal nerves.3,4 To test this suggestion we recorded the motor-unit action potential associated with the anal reflex, since damaged pudendal nerves might show abnormalities of latency, amplitude, or duration of the reflex response. We describe here a simple electrophysiological method for recording the anal reflex in man, and present preliminary data on its latency in 13 normal subjects (aged 22-75 yr, mean 43), 10 of whom were female, and in patients with various disorders affecting the pelvic-floor musculature. Since such disorders are commoner in women than in men, we have deliberately studied more women than men. Methods The subjects were placed in the left lateral position. A con-
1291 centric needle E.M.G. electrode was inserted into the superficial portion of the external anal sphincter muscle in the midline
position posterior to the anal verge, and the perianal skin was stimulated by a bipolar surface electrode (standard ’DISA 14A21’ E.M.G. apparatus). A ground electrode was applied to the thigh. The reflexly evoked muscle action potentials were displayed on the oscilloscope of the E.M.G. apparatus or on a separate ’Telequipment DM 64’ storage oscilloscope. Squarewave stimuli of 0-1 1 to 0-2 ms duration and of variable voltage were used. The reflexly evoked electrical responses detected by the needle electrode in the external anal sphincter muscle were generally maximum with stimuli of about 90 V.
Results’ The muscle action potential in a normal anal reflex recorded the oscilloscope (fig. 1) coincided with contraction of the external anal sphincter muscle, recognised by transient constriction of the anal margin. The latency of the electrical response in this record was 7.9 ms. The ranges and mean values of the latencies of the anal reflex in normal subjects are as follows : on
There
changes in of the responses
were no
amplitudes
latency ranged
with increasing age. The from 50 to 500 fLV (mean
125V). The reflex response to a single stimulus is usually readily recognisable (fig. 1), but the random basal activity in the normal external anal sphincter5 may sometimes make identification of the reflexly evoked muscle action potential difficult. In some patients the reflex response is of such low amplitude that it approximates to that of basal contractions. Repeated supramaximum stimuli (usually about 90 V), delivered at about 1/s, can then be used to evoke a reflex response with a conspicuous,
latency, which can be readily isolated from basal contractions of the sphincter muscle (fig. 2). Both the interval between stimulus and response and the form of the response suggest that the latter is not an artefact resulting from direct spread of current from the stimulating to the recording electrode. However, since the two electrodes were only about 6 cm apart, we tested this possibility. In one patient, therefore, we infiltrated the skin underlying the stimulating electrode with 2 ml of 1% xylocaine. This produced an area of complete anaesthesia under the stimulating electrode at the anal margin and abolished the reflex response for about 30 min. The basal potentials, which were of much smaller amplitude than the reflex itself, were not affected by the local anoesthesia. A similar injection of normal saline, at a later date, did not alter the reflex response. The latency of the response was constant when supramaximum stimuli were used (fig. 2), and this was also the shortest latency recorded in each subject. Stimuli of lesser amplitude produced longer latencies. That the latency cannot be reduced further was confirmed in 1 subject by measuring it immediately after a supramaximum tetanus had been applied to the stimulation point on the perianal skin. No change in latency resulted after this procedure.
constant
Discussion This
simple electrophysiological method for studying the of the anal reflex, and the amplitude and duration of the response, gives reproducible results in individual subjects. It is not uncomfortable if the perianal cutaneous stimuli are applied less frequently than about 1/s. The afferent pathways of the anal reflex lie in the perianal branch of the pudendal nerves, which synapse in S2 and S3 segments of the spinal cord,’’6’’and the efferent pathway in the pudendal nerves and their perineal and inferior rectal branches.4,6 The response is detected in the external anal sphincter muscles and probably also the puborectalis and part of the levator ani muscles.6 Since the anal reflex, like the abdominal reflex,8 is evoked by cutaneous stimulation, it is probably polysynaptic. The period of central synaptic delay in polysynaptic reflexes is variable. When recording the latency of the anal reflex it is important, therefore, to establish the shortest possible latency which can be done by ensuring that the cutaneous stimulus is supramaximum, since prior tetanisation of the perianal skin did not shorten the latency. The reflex mechanisms underlying faecal continence are of fundamental importance but they have been little studied since the anorectal balloon pressure recordings of Denny-Brown and Robertson.9 A recording of the anal reflex in a patient with idiopathic faecal incontinence shows that the latency of the reflex was substantially increased (20 ms) and its amplitude (20 fL V) was less than that of normal subjects (fig. 2). These observations suggest that electrophysiological recording of the anal reflex in man may prove useful in assessing the function of the nerves supplying the muscles of the pelvic floor, especially in patients with fsecal incontinence and with descending perineum syndrome in whom stretch-induced injury to the pudendal nerves, perhaps associated with entrapment,3 might lead to detectable changes in latency, amplitude, or duration of the evoked response.
latency
We thank Sir Alan Parks for his encouragement. M. M. H. is sup-
ported by the St. Mark’s Hospital Research Foundation. REFERENCES
1. Aminev, A. M., Amineva, V. A. J. Procrol. 1969, 20, 339. 2. Walton, J. N. in Brain’s Diseases of the Nervous System;
Fig. 2-Anorectal incontinence. Arrow indicates anal reflex
response at
a constant
1/s). Other complexes are "basal" potentials Latency in this case is 19.2ms.
latency (stimuli sphincter.
of anal
1977. 3. Parks, A. G., Swash, M., Urich, H. Gut, 1977, 18, 656. 4. Beersiek, F., Swash, M. Unpublished. 5. Floyd, W. F., Walls, E. W. J. Physiol. 1953, 122, 599. 6. Bishop, B. Neurophysiology, 1959, 22, 679. 7. Sherrington, C. S. J. Physiol. 1892, 13, 672. 8. Kugelberg, E., Hagbarth, K. E. Brain, 1958, 81, 290. 9. Denny-Brown, D., Robertson, G. ibid, 1935, 58, 256.
p. 751. Oxford.
