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Localization of substance P and enkephalin by immunohistochemistry in the spinal cord of human fetus
0306-4522/88$3.00+ 0.00 Pergamon Press plc IBRO
Neuroscience Vol. 27, No. 3, pp. 989-993, 1988 Printed in Great Britain
LOCALIZATION OF SUBSTANCE P AND ENKEPHALIN BY IMMUNOHISTOCHEMISTRY IN THE SPINAL CORD OF HUMAN FETUS *Department
C. B. Luo,* D. R. ZHENG,* Y. L. GUAN* and D. T. YEW? of Anatomy, Jinan University, Guangzhou, China and TDepartment of Anatomy, The Chinese University of Hong Kong, Shatin, Hong Kong
Abstract-The peroxidase-antiperoxidase method was used to study the distribution of substance P and enkephalin during development of the spinal cords of human fetuses. Thirty-seven cases were collected, ranging from 5- to 40-weeks-old (fetal ages). Both types of transmitters were present initially around the fifth week in the mantle layer of the base of the dorsal horn, around the tenth week at the anterior gray and the intermediate gray and around the sixth week at the marginal layer at the base of the ventrolateral funiculus. Substance P- and enkephalin-positive sites at the marginal layers at the base of the dorsolateral funiculus were evident in the same area at 5-6 weeks. The positive fibers in the dorsal horn were initially located in the superficial layers. By the eleventh week, the positive sites spread to other surface layers at the lateral sides of the dorsal horns bilaterally at all spinal levels above the sacral. In the sacral levels adjacent to the conus medullaris, the spreading to surface layers was not apparent bilaterally until the seventeenth week. By weeks 18-26 the positive sites penetrated deeper in the dorsal horn and by week 27 assumed the adult path. The enkephalin cell bodies were present in the Rexed layers I and II of the dorsal horn and the substance P-nositive sites were apparent in the dorsal ganglia of the 28&40-week-old
During the past decade, interests have been centered around the positive role of substance P (SP) and enkephalin (ENK) as transmitters for pain sensation or modification. The majority of these studies utilized animal models and reports on humans were rare, e.g. works of Charnay et al. 3.4 Even in these reports, human data on fetuses prior to the tenth week were absent. This piece of work attempts to further clarify this dilemma. EXPERIMENTAL PROCEDURES Thirty-seven dead fetuses were used ranging from fetal ages of 5 to 40 weeks. Fetal ages were computed from ages of conception and the crown-rump lengths (Table 1). The fetuses were taken out 1-3 h after death. They were perfused through the hearts with 4% paraformaldehyde in phosphate buffer (PH 7.2). Cervical, thoracic, lumbar and sacral segments of the spinal cords as well as the dorsal root ganglia were dissected out and again postfixed in the same solution. They were then washed with 20% sucrose phosphate-buffered saline (PBS) and cut with a cryostat at 2540 urn. Localization of the substance P and ENK was performed according to the peroxidase-antiperoxidase (PAP) method.14 The sections were put in solutions containing 0.25% Triton X-100 solution with 3% calf serum. They were subsequently transferred to rabbit anti-substance P serum or rabbit anti-enkephalin serum (dilution I :6000, Peninsular, U.S.A.) at 37°C for 3 h, followed by 4°C for 48-60 h. They were then reacted with goat anti-rabbit serum for 1 h followed by reaction with PAP complex (dilution 1:30, 1st Shanghai Medical School & Wuhan Medical
Abbreviations: ENK, enkephalin; PAP, antiperoxidase; PBS, phosphate-buffered substance P.
peroxidasesaline; SP,
University) at 37°C for 30min followed by treatment with 3.3’-diaminobenzidine (0.05%) ~1~s 0.01% H,O, and 0105M Tris-HCl. In between the steps, washing wiih 0.01 M PBS was performed. Blank and adsorption controls were carried out to ensure the antigen specificity. SP and LeuENK antigens were supplied by Sigma, U.S.A. RESULTS
In general, the positive immunological sites were mainly in the processes and terminals and took the forms of brownish varicosities. At some ages, in the dorsal horn and the dorsal ganglia, there were also positive cell bodies. Dorsal horn
SP- and ENK-positive sites were initially present in the mantle layer at the base of the dorsal horns in the fifth-week fetus (Figs 1 and 2) and the intensity of the reaction increased by fetal weeks 6-10. By the eleventh week, in the lateral regions of the Rexed layers I and II and all spinal levels above the sacrum, there were concentrations of positive fibers bilaterally (Fig. 3). At the lower spinal levels, i.e. sacrum near the conus medullaris, the concentrations of positive fibers in the Rexed layers I and II did not appear until the fourteenth week and bilateral concentration was not evident until weeks 15-17. The positive reaction in the lateral portion of the dorsal horn was significantly greater than in the medial portion. In general, the immunohistochemical ENK activities were not as significant as those of substance P. By weeks 18-26, three groups of positive fibers (both SP 989
C. B. Luo et al.
990
Table 1. Fetal ages and crown-rump Fetal age (weeks) Crown-rump (mm)
5
6
7
8
Crown-rump length (mm) Number of cases
1
4
2
5
1
164
197
208
215
1
1
1
1
250 253 2
and ENK) started to penetrate deeper (Figs 4 and 5); they were (1) a lateral group which had the strongest reaction and entered the marginal areas of the dorsal
horn surrounding the substantia gelatinosa into the deep layers; (2) an intermediate group (l-2 bundles) less reactive
intensity
and
which
eventually
united with the penetrating lateral group and reached the reticular nucleus, nuclei intermediolateral and intermediomedial, Clarke’s nucleus as well as the nucleus of the posterior commissure; and (3) a medial group with the least positivity. This last group followed the boundary between the inner margin of the dorsal horn and the posterior funiculus to the nucleus of the posterior commissure with a few side branches to the posterior funiculus, the periaqueductal gray, and occasionally as far as the nucleus of the anterior commissures. Positive ENK cell bodies initially appeared by week 26 at dorsal horn layers I and II (Fig. 6) and persisted until week 28. By week 27, the distribution of immunopositive fibers and terminals in the dorsal horn represented the adult pattern, with a few fibers extending to the deep aspects of the anterior horn (Fig. 7). SP-positive cell bodies, however, were absent throughout.
lengths
11
12
13
14
15
17
110 112
117
2
1
125 127 142 3
150 156
97 1
100 105 110 3
256
287
295
330
332
335
1
1
1
1
1
1
length
Number of cases
with
10
2
The intermediate gray and the reticular nucleus Positive fibers (SP and ENK) were scattered in these areas by week 10 in the cervical, thoracic and lumbar spinal levels. SP positivities were particularly
significant (Fig. 8). By week 11, larger fibers with bead-like appearance were seen. For the sacral regions adjacent to the conus medullaris, the two types of positive fibers were absent before week 13. Positive sites (both types) in Clark’s nucleus were initially detected by the tenth week. By weeks 18-26, the positive fibers (both types) surrounding Clarke’s nucleus were significant but the positive sites (both types) inside the nucleus decreased (Fig. 9). By week 27, the pattern was the same as in the adult human. Anterior gray
Both types of immunohistochemical positivity appeared initially by the tenth week in the various areas of the spinal cord (Table 2), and increased in intensity by weeks 11-17. By weeks 18-26, the positive sites remained scattered but positive fibers had penetrated
into the anterior gray mainly from the anterior funiculus. After week 27, fibers were present in both the anterior and posterior gray but those in the
Fig. 1. Fetal age 5 weeks. Arrows point to mantle layer at the base of the dorsal horn of spinal cord showing inconspicuous SP-positive sites at low magnification (x 33) which become obvious at high magnification ( x 120, inset). Thoracic level. Fig. 2. Fetal age 6 weeks. Arrows point to ENK-positive sites at the base of the dorsal horn of the spinal cord. Note they are only conspicuous at high magnification ( x 120). Lumbar level. Fig. 3. Fetal age 11 weeks. Arrow points to the lateral regions of superficial layers of dorsal horn which show concentration of SP-positive sites and which are obvious even under low magnification (x 33). Cervical level. Figs 4 and 5. Fetal week 26. Superficial layers of dorsal horn showing medial (F,), intermediate (F,) and lateral bundles (F,) penetrating deeper as ENK-positive fibers (Fig. 4) and SP-positive fibers (Fig. 5). Figure 5 was counterstained with Cresyl Violet. Note reactive products are more obvious in the SP reactions ( x 120). Cervical level. Fig. 6. Fetal week 26. Arrows point to the ENK-positive sites in the Rexed layer II which are not obvious under low magnification (x 33) but become obvious at x 120 in both cell bodies and processes (inset). CC, central canal. Cervical level. Fig. 7. Fetal age 28 weeks. Arrows point to ENK-positive fibers penetrating from the dorsal horn into the anterior horn region even at low magnification (x 33). CC, central canal; FMA, anterior median fissure. Thoracic level. Fig. 8. Fetal
week 10. Arrows point to SP-positive sites in the intermediolateral reticular nucleus (NR) ( x 120). Thoracic level.
nucleus
(MNL)
and
Fig. 9. Fetal week 28. CL, positive ENK fibers surrounding Clarke’s nucleus. Arrow denotes few ENK positive sites inside Clarke’s nucleus itself. sm and SC represent layers 1 and II of dorsal horn (x 33). Thoracic level. Fig. 10. Fetal week 40. Arrows
point to SP-positive cell bodies and processes Cervical level.
in dorsal
ganglion
( x 120).
Substance P and enkephalin in human fetal spinal cord anterior compared
gray appeared
to decrease
in intensity when
with early stages.
White matter SP varicosities initially were present in the dorsolateral region of the marginal layer by the fifth week. However, ENK-positive varicosities were absent until the sixth week in the same area. By the tenth week, both the anterior and posterior funiculus were scattered with these two types of positive fibers.
991
Dorsal ganglion Initially SP-positive neurons were present by week 28 (14-16 per section). By week 40, the number of neurons increased (26-28 per section) (Fig. 10). ENK activities were totally absent. DISCUSSION
Our results point out that as early as the fifth week, a few SP-positive varicosities were apparent at the
Figs I-10.
C. B. Lu0 et al.
992
Table 2. Sites of initial appearances spinal cords Fetal age (weeks) 5
of SP and ENK fibers and terminals and their fetal ages
SP fibers and terminals mantle layer (base of dorsal horn) marginal layer (base of posterolateral funiculus)
6
10
dorsolateral mantle and marginal areas of the spinal cords. According to Cochard et al.,5 when the sympathetic ganglia were initially formed by aggregation of neurons in the 11.5-day rat embryo, they already demonstrated noradrenaline positivities. This shows that neurochemicals may be present prior to distinct morphogenesis. In the human, Warwick and Williams” described the presence of neurofibrils in the marginal layer of the spinal cord as early as the fourth week and these fibrils appear to arise from the neurons of the mantle layer as well as from those of the dorsal ganglion. Kessler and BlacklO analysed the SP quantity in the dorsal ganglion and the posterior part of the spinal cord and discovered the presence of SP in both regions on the same day of pregnancy. It is therefore possible that in the human fetus, between the dorsal horn and the dorsal ganglion, there exist fiber connections which are probably SP fibers. Kessler and Black” suggest that perhaps the SP-positive neurons are already present in the neural crests before migration to form the dorsal ganglia, although they may contain only such minute quantities of SP that it is not detectable. Our results indicate that by the fifth week, a time when the neural tube initially closes and the dorsal ganglion is just formed by the crest cells, SP positivity was already registering. Our results, therefore, tend to agree with Kessler and Black’s suggestions. Pickel er nl.13 reported the presence of SP and ENK positivity by days 15-18 of pregnancy in the dorsal horn of the rat. Our results indicate the initial presence of both types of activities in the dorsolateral mantle layer of human fetus at a much earlier stage of pregnancy, unlike those reported by Pickel et ~1.‘~in the rat. On the other hand, they have reported on the presence of positive SP and ENK neurons in the spinal cord of the rat; we have failed to locate any SP neurons in the spinal cord of our human fetuses. Charnay et al.3,4 indicated that ENK-positive fibers are present around the tenth week in the dorsolateral funiculus of the spinal cord
in the
ENK fibers and terminals mantle layer (base of dorsal horn)
marginal layer (base of posterolateral and anterolateral funiculi) substantia gelatinosa (II) nucleus proprius (III) IV, V, VI layers Clarke’s nucleus intermediate grey VII reticular nucleus anterior grey VIII, IX periaqueductal X anterior and posterior funiculi
during human development. The initial appearance of SP and ENK fibers inside the dorsal horn is shown later. Their report on the SP and ENK presence in the anterior horn shows even later, appearing at week 16. This is considerably later than that of this study. For example, from our results, SP fibers are present in the anterior horn, intermediate gray and the reticular nucleus by the tenth week. These discrepancies may be due to different methods of labeling, the time lapses in the collection of specimens and the availability of the specimens. Charnay et ~1.~3~utilized immunofluorescence and collection of their specimens was sometimes delayed until 24 h after death. In their ENK specimens, the lowest fetal age was 10 weeks (one case) and in their SP specimens, the lowest fetal age was 12 weeks (two cases). SP and ENK appear to concentrate in the dorsal horn.‘%*,’Our work indicates that in the dorsal horn, the distribution of reactive sites starts to concentrate in the course of development. Initial increase of activity and concentration of activity appears by about the eleventh week, this possibly related to the rapid growth of the limbsI In one experiment, Kessler and BlacklO removed the anterior limb of the rat at day 17 of pregnancy and found a decrease in concentration of SP reactivity, indicating that target influence is important. In another experiment, Linda et al.” reported the presence of opiate receptors in the SP efferents in the substantia gelatinosa and sectioning of the dorsal root affected the binding activity of the opiate compounds. Conversely, injection of morphine would potentiate the immunoreactivity of SP. Indeed, complicated relationships seem to exist between the targets and the development of these neurotransmitters in the CNS. Our results further indicate the late presence of SP and ENK transmitters in the sacral regions of the spinal cord. As these regions are near to the conus medullaris and closing of the neural tube in this area of the spinal cord comes last, it appears again that a
Substance
P and enkephalin
close relationship between morphogenic development and the appearance of nemotransmitters may exist. In fact it has been proposed by Nordes and Dasi2 that SP and ENK transmitters are originated from the neural tube itself. The reason why they appear at different ages is related to the development of the neuronal system as well as the projection of the terminals. Our observations also show that the SP-positive neurons in the spinal ganglia initially appear by week 28 and increase in number as the fetus ages. Kessler and Black” regard this as a reflection of neuron maturation. Many of the SP fibers and terminals in the posterior regions of the spinal cord are actually projections from the SP-positive neurons in the dorsal ganglion. Thus, the development of the spinal cord correlates with the maturation of these SP neurons inside the dorsal ganglia.
in human
fetal spinal cord
993
Of further interest is the presence of ENK-positive neurons in the substantia gelatinosa at fetal ages of 2628 weeks. De Lanerolle and La Motte6 documented that in layers II and III of the dorsal horn, there are islet cells and stalk cells which receive SP efferents. Hunt et cd9 further suggest that amongst the islet cells there are positive ENK neurons with ENK varicosities in their dendritic system. Aronin et al.,’ using electron microscopy, confirm the presence of ENK-positive dendrites in the substantia gelatinosa of the spinal cord. Gobel et ~2.’ regard that the islet cells and the stalked cells are, respectively, excitatory and inhibitory ENK neurons. This integration of SP and ENK components in the spinal cord, present in the mid to later periods of pregnancy, demonstrate that by this time, the fetus has all the anatomical and neurochemical features required to receive pain and traumatic sensations.
REFERENCES
1. 2. 3. 4. 5. 6. I. 8.
9.
Aronin N., Difighia M., Liotta A. S. and Martin J. B. (1981) Ultrastructural
localization and biochemical features of immunoreactive leu-enkephalin in monkey dorsal horn. J. Neurosci. 1, 561-577. Barber R. P., Vaughn J. E., Slemmon J. R., Salvaterra P. M., Roberts E. and Leeman S. E. (1979) The origin, distribution and synaptic relationships of substance P axons in rat spinal cord. J. contp. Neural. 18.4, 331-352. Charnay Y., Paulin C., Chayviallet J. A. and Dubois P. M. (1983) Distribution of substance P-like immunoreactivity in the spinal cord and dorsal root ganglia of the human foetus and infant. Neuroscience 10, 41-55. Charnay Y., Paulin C., Dray F. and DuBois P. M. (1984) Distribution of enkephahn in human fetus and infant spinal cord: an immunofluorescence study. J. romp. Neurol. 223, 415423. C&hard P., Goldstein M. and Black I. B. (1979) Initial development of the noradrenergic phenotype in autonomic neuroblasts of the rat embryo in vivo. Devl Biol. 71, 100-114. De Lanerolle N. C. and La Motte C. C. (1982) The human spinal cord: substance P and Met-enkephahn immunoreactivity. J. Neurosci. 2, 1369-1386. Glazer E. J. and Basbaum A. I. (1981) Immunohistochemical localization of leucine-enkephalin in the spinal cord of the cat: enkephahn-containing marginal neurons and pain modulation. J. camp. Neural. 196, 377-390. Gobel S., Falls W. M., Bennett G. J., Abdelmoumene M., Hayashi H. and Humphrey E. (1980) An EM analysis of the synaptic connections of horseradish peroxidase-filled stalked cells and islet cells in the substantia gelatinosa of adult cat spinal cord. J. camp. Neural. 194, 781-808.
Hunt S. P., Kelly J. S. and Emson P. C. (1980) The electron microscopic localizations of methionine-enkephalin within the superticial layers (I and II) of the spinal cord. Neuroscience 5, 1871-1902.
10. Kessler J. A. and Black I. B. (1981) Nerve growth factor stimulates development of substance P in the embryonic spinal cord. Brain Res. 208, 135-145. S. J. and Naffchi N. E. (1980) Effect of morphine on substance P neurons in rat spinal cord, II. Linda L. V., Abrahams preliminary study. Brain Res. 182, 229-236. 12. Nordes H. 0. and Das G. P. (1973) Temporal patterns of neurogenesis in the spinal cord of rat. 1. An autoradiographic study-time and sites of origin and migration and settling patterns of neuroblasts. Bruin Res. 73, 121-138. of substance P- and enkephalin13. Pickel V. M., Sumal K. K. and Miller R. J. (1982) Early prenatal development containing neurons in the rat. J. camp. Neural. 210, 411422. L. A. (1979) The unlabelled antibody peroxidase-antiperoxidase (PAP) method. In Immunocytochemistry 14. Sternberger (ed. Sternberger L.), pp. 104169. John Wiley, New York. Edinburgh. 15. Warwick R. and Williams P. D. (1973) Gray’s Anaromy, 3rd edn, pp. 85, 129, 197. Churchill Livingstone, (Accepted
22 June 1988)
Neuroscience Vol. 27, No. 3, pp. 989-993, 1988 Printed in Great Britain
LOCALIZATION OF SUBSTANCE P AND ENKEPHALIN BY IMMUNOHISTOCHEMISTRY IN THE SPINAL CORD OF HUMAN FETUS *Department
C. B. Luo,* D. R. ZHENG,* Y. L. GUAN* and D. T. YEW? of Anatomy, Jinan University, Guangzhou, China and TDepartment of Anatomy, The Chinese University of Hong Kong, Shatin, Hong Kong
Abstract-The peroxidase-antiperoxidase method was used to study the distribution of substance P and enkephalin during development of the spinal cords of human fetuses. Thirty-seven cases were collected, ranging from 5- to 40-weeks-old (fetal ages). Both types of transmitters were present initially around the fifth week in the mantle layer of the base of the dorsal horn, around the tenth week at the anterior gray and the intermediate gray and around the sixth week at the marginal layer at the base of the ventrolateral funiculus. Substance P- and enkephalin-positive sites at the marginal layers at the base of the dorsolateral funiculus were evident in the same area at 5-6 weeks. The positive fibers in the dorsal horn were initially located in the superficial layers. By the eleventh week, the positive sites spread to other surface layers at the lateral sides of the dorsal horns bilaterally at all spinal levels above the sacral. In the sacral levels adjacent to the conus medullaris, the spreading to surface layers was not apparent bilaterally until the seventeenth week. By weeks 18-26 the positive sites penetrated deeper in the dorsal horn and by week 27 assumed the adult path. The enkephalin cell bodies were present in the Rexed layers I and II of the dorsal horn and the substance P-nositive sites were apparent in the dorsal ganglia of the 28&40-week-old
During the past decade, interests have been centered around the positive role of substance P (SP) and enkephalin (ENK) as transmitters for pain sensation or modification. The majority of these studies utilized animal models and reports on humans were rare, e.g. works of Charnay et al. 3.4 Even in these reports, human data on fetuses prior to the tenth week were absent. This piece of work attempts to further clarify this dilemma. EXPERIMENTAL PROCEDURES Thirty-seven dead fetuses were used ranging from fetal ages of 5 to 40 weeks. Fetal ages were computed from ages of conception and the crown-rump lengths (Table 1). The fetuses were taken out 1-3 h after death. They were perfused through the hearts with 4% paraformaldehyde in phosphate buffer (PH 7.2). Cervical, thoracic, lumbar and sacral segments of the spinal cords as well as the dorsal root ganglia were dissected out and again postfixed in the same solution. They were then washed with 20% sucrose phosphate-buffered saline (PBS) and cut with a cryostat at 2540 urn. Localization of the substance P and ENK was performed according to the peroxidase-antiperoxidase (PAP) method.14 The sections were put in solutions containing 0.25% Triton X-100 solution with 3% calf serum. They were subsequently transferred to rabbit anti-substance P serum or rabbit anti-enkephalin serum (dilution I :6000, Peninsular, U.S.A.) at 37°C for 3 h, followed by 4°C for 48-60 h. They were then reacted with goat anti-rabbit serum for 1 h followed by reaction with PAP complex (dilution 1:30, 1st Shanghai Medical School & Wuhan Medical
Abbreviations: ENK, enkephalin; PAP, antiperoxidase; PBS, phosphate-buffered substance P.
peroxidasesaline; SP,
University) at 37°C for 30min followed by treatment with 3.3’-diaminobenzidine (0.05%) ~1~s 0.01% H,O, and 0105M Tris-HCl. In between the steps, washing wiih 0.01 M PBS was performed. Blank and adsorption controls were carried out to ensure the antigen specificity. SP and LeuENK antigens were supplied by Sigma, U.S.A. RESULTS
In general, the positive immunological sites were mainly in the processes and terminals and took the forms of brownish varicosities. At some ages, in the dorsal horn and the dorsal ganglia, there were also positive cell bodies. Dorsal horn
SP- and ENK-positive sites were initially present in the mantle layer at the base of the dorsal horns in the fifth-week fetus (Figs 1 and 2) and the intensity of the reaction increased by fetal weeks 6-10. By the eleventh week, in the lateral regions of the Rexed layers I and II and all spinal levels above the sacrum, there were concentrations of positive fibers bilaterally (Fig. 3). At the lower spinal levels, i.e. sacrum near the conus medullaris, the concentrations of positive fibers in the Rexed layers I and II did not appear until the fourteenth week and bilateral concentration was not evident until weeks 15-17. The positive reaction in the lateral portion of the dorsal horn was significantly greater than in the medial portion. In general, the immunohistochemical ENK activities were not as significant as those of substance P. By weeks 18-26, three groups of positive fibers (both SP 989
C. B. Luo et al.
990
Table 1. Fetal ages and crown-rump Fetal age (weeks) Crown-rump (mm)
5
6
7
8
Crown-rump length (mm) Number of cases
1
4
2
5
1
164
197
208
215
1
1
1
1
250 253 2
and ENK) started to penetrate deeper (Figs 4 and 5); they were (1) a lateral group which had the strongest reaction and entered the marginal areas of the dorsal
horn surrounding the substantia gelatinosa into the deep layers; (2) an intermediate group (l-2 bundles) less reactive
intensity
and
which
eventually
united with the penetrating lateral group and reached the reticular nucleus, nuclei intermediolateral and intermediomedial, Clarke’s nucleus as well as the nucleus of the posterior commissure; and (3) a medial group with the least positivity. This last group followed the boundary between the inner margin of the dorsal horn and the posterior funiculus to the nucleus of the posterior commissure with a few side branches to the posterior funiculus, the periaqueductal gray, and occasionally as far as the nucleus of the anterior commissures. Positive ENK cell bodies initially appeared by week 26 at dorsal horn layers I and II (Fig. 6) and persisted until week 28. By week 27, the distribution of immunopositive fibers and terminals in the dorsal horn represented the adult pattern, with a few fibers extending to the deep aspects of the anterior horn (Fig. 7). SP-positive cell bodies, however, were absent throughout.
lengths
11
12
13
14
15
17
110 112
117
2
1
125 127 142 3
150 156
97 1
100 105 110 3
256
287
295
330
332
335
1
1
1
1
1
1
length
Number of cases
with
10
2
The intermediate gray and the reticular nucleus Positive fibers (SP and ENK) were scattered in these areas by week 10 in the cervical, thoracic and lumbar spinal levels. SP positivities were particularly
significant (Fig. 8). By week 11, larger fibers with bead-like appearance were seen. For the sacral regions adjacent to the conus medullaris, the two types of positive fibers were absent before week 13. Positive sites (both types) in Clark’s nucleus were initially detected by the tenth week. By weeks 18-26, the positive fibers (both types) surrounding Clarke’s nucleus were significant but the positive sites (both types) inside the nucleus decreased (Fig. 9). By week 27, the pattern was the same as in the adult human. Anterior gray
Both types of immunohistochemical positivity appeared initially by the tenth week in the various areas of the spinal cord (Table 2), and increased in intensity by weeks 11-17. By weeks 18-26, the positive sites remained scattered but positive fibers had penetrated
into the anterior gray mainly from the anterior funiculus. After week 27, fibers were present in both the anterior and posterior gray but those in the
Fig. 1. Fetal age 5 weeks. Arrows point to mantle layer at the base of the dorsal horn of spinal cord showing inconspicuous SP-positive sites at low magnification (x 33) which become obvious at high magnification ( x 120, inset). Thoracic level. Fig. 2. Fetal age 6 weeks. Arrows point to ENK-positive sites at the base of the dorsal horn of the spinal cord. Note they are only conspicuous at high magnification ( x 120). Lumbar level. Fig. 3. Fetal age 11 weeks. Arrow points to the lateral regions of superficial layers of dorsal horn which show concentration of SP-positive sites and which are obvious even under low magnification (x 33). Cervical level. Figs 4 and 5. Fetal week 26. Superficial layers of dorsal horn showing medial (F,), intermediate (F,) and lateral bundles (F,) penetrating deeper as ENK-positive fibers (Fig. 4) and SP-positive fibers (Fig. 5). Figure 5 was counterstained with Cresyl Violet. Note reactive products are more obvious in the SP reactions ( x 120). Cervical level. Fig. 6. Fetal week 26. Arrows point to the ENK-positive sites in the Rexed layer II which are not obvious under low magnification (x 33) but become obvious at x 120 in both cell bodies and processes (inset). CC, central canal. Cervical level. Fig. 7. Fetal age 28 weeks. Arrows point to ENK-positive fibers penetrating from the dorsal horn into the anterior horn region even at low magnification (x 33). CC, central canal; FMA, anterior median fissure. Thoracic level. Fig. 8. Fetal
week 10. Arrows point to SP-positive sites in the intermediolateral reticular nucleus (NR) ( x 120). Thoracic level.
nucleus
(MNL)
and
Fig. 9. Fetal week 28. CL, positive ENK fibers surrounding Clarke’s nucleus. Arrow denotes few ENK positive sites inside Clarke’s nucleus itself. sm and SC represent layers 1 and II of dorsal horn (x 33). Thoracic level. Fig. 10. Fetal week 40. Arrows
point to SP-positive cell bodies and processes Cervical level.
in dorsal
ganglion
( x 120).
Substance P and enkephalin in human fetal spinal cord anterior compared
gray appeared
to decrease
in intensity when
with early stages.
White matter SP varicosities initially were present in the dorsolateral region of the marginal layer by the fifth week. However, ENK-positive varicosities were absent until the sixth week in the same area. By the tenth week, both the anterior and posterior funiculus were scattered with these two types of positive fibers.
991
Dorsal ganglion Initially SP-positive neurons were present by week 28 (14-16 per section). By week 40, the number of neurons increased (26-28 per section) (Fig. 10). ENK activities were totally absent. DISCUSSION
Our results point out that as early as the fifth week, a few SP-positive varicosities were apparent at the
Figs I-10.
C. B. Lu0 et al.
992
Table 2. Sites of initial appearances spinal cords Fetal age (weeks) 5
of SP and ENK fibers and terminals and their fetal ages
SP fibers and terminals mantle layer (base of dorsal horn) marginal layer (base of posterolateral funiculus)
6
10
dorsolateral mantle and marginal areas of the spinal cords. According to Cochard et al.,5 when the sympathetic ganglia were initially formed by aggregation of neurons in the 11.5-day rat embryo, they already demonstrated noradrenaline positivities. This shows that neurochemicals may be present prior to distinct morphogenesis. In the human, Warwick and Williams” described the presence of neurofibrils in the marginal layer of the spinal cord as early as the fourth week and these fibrils appear to arise from the neurons of the mantle layer as well as from those of the dorsal ganglion. Kessler and BlacklO analysed the SP quantity in the dorsal ganglion and the posterior part of the spinal cord and discovered the presence of SP in both regions on the same day of pregnancy. It is therefore possible that in the human fetus, between the dorsal horn and the dorsal ganglion, there exist fiber connections which are probably SP fibers. Kessler and Black” suggest that perhaps the SP-positive neurons are already present in the neural crests before migration to form the dorsal ganglia, although they may contain only such minute quantities of SP that it is not detectable. Our results indicate that by the fifth week, a time when the neural tube initially closes and the dorsal ganglion is just formed by the crest cells, SP positivity was already registering. Our results, therefore, tend to agree with Kessler and Black’s suggestions. Pickel er nl.13 reported the presence of SP and ENK positivity by days 15-18 of pregnancy in the dorsal horn of the rat. Our results indicate the initial presence of both types of activities in the dorsolateral mantle layer of human fetus at a much earlier stage of pregnancy, unlike those reported by Pickel et ~1.‘~in the rat. On the other hand, they have reported on the presence of positive SP and ENK neurons in the spinal cord of the rat; we have failed to locate any SP neurons in the spinal cord of our human fetuses. Charnay et al.3,4 indicated that ENK-positive fibers are present around the tenth week in the dorsolateral funiculus of the spinal cord
in the
ENK fibers and terminals mantle layer (base of dorsal horn)
marginal layer (base of posterolateral and anterolateral funiculi) substantia gelatinosa (II) nucleus proprius (III) IV, V, VI layers Clarke’s nucleus intermediate grey VII reticular nucleus anterior grey VIII, IX periaqueductal X anterior and posterior funiculi
during human development. The initial appearance of SP and ENK fibers inside the dorsal horn is shown later. Their report on the SP and ENK presence in the anterior horn shows even later, appearing at week 16. This is considerably later than that of this study. For example, from our results, SP fibers are present in the anterior horn, intermediate gray and the reticular nucleus by the tenth week. These discrepancies may be due to different methods of labeling, the time lapses in the collection of specimens and the availability of the specimens. Charnay et ~1.~3~utilized immunofluorescence and collection of their specimens was sometimes delayed until 24 h after death. In their ENK specimens, the lowest fetal age was 10 weeks (one case) and in their SP specimens, the lowest fetal age was 12 weeks (two cases). SP and ENK appear to concentrate in the dorsal horn.‘%*,’Our work indicates that in the dorsal horn, the distribution of reactive sites starts to concentrate in the course of development. Initial increase of activity and concentration of activity appears by about the eleventh week, this possibly related to the rapid growth of the limbsI In one experiment, Kessler and BlacklO removed the anterior limb of the rat at day 17 of pregnancy and found a decrease in concentration of SP reactivity, indicating that target influence is important. In another experiment, Linda et al.” reported the presence of opiate receptors in the SP efferents in the substantia gelatinosa and sectioning of the dorsal root affected the binding activity of the opiate compounds. Conversely, injection of morphine would potentiate the immunoreactivity of SP. Indeed, complicated relationships seem to exist between the targets and the development of these neurotransmitters in the CNS. Our results further indicate the late presence of SP and ENK transmitters in the sacral regions of the spinal cord. As these regions are near to the conus medullaris and closing of the neural tube in this area of the spinal cord comes last, it appears again that a
Substance
P and enkephalin
close relationship between morphogenic development and the appearance of nemotransmitters may exist. In fact it has been proposed by Nordes and Dasi2 that SP and ENK transmitters are originated from the neural tube itself. The reason why they appear at different ages is related to the development of the neuronal system as well as the projection of the terminals. Our observations also show that the SP-positive neurons in the spinal ganglia initially appear by week 28 and increase in number as the fetus ages. Kessler and Black” regard this as a reflection of neuron maturation. Many of the SP fibers and terminals in the posterior regions of the spinal cord are actually projections from the SP-positive neurons in the dorsal ganglion. Thus, the development of the spinal cord correlates with the maturation of these SP neurons inside the dorsal ganglia.
in human
fetal spinal cord
993
Of further interest is the presence of ENK-positive neurons in the substantia gelatinosa at fetal ages of 2628 weeks. De Lanerolle and La Motte6 documented that in layers II and III of the dorsal horn, there are islet cells and stalk cells which receive SP efferents. Hunt et cd9 further suggest that amongst the islet cells there are positive ENK neurons with ENK varicosities in their dendritic system. Aronin et al.,’ using electron microscopy, confirm the presence of ENK-positive dendrites in the substantia gelatinosa of the spinal cord. Gobel et ~2.’ regard that the islet cells and the stalked cells are, respectively, excitatory and inhibitory ENK neurons. This integration of SP and ENK components in the spinal cord, present in the mid to later periods of pregnancy, demonstrate that by this time, the fetus has all the anatomical and neurochemical features required to receive pain and traumatic sensations.
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