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Journal of the Anatomical Society of India

Histogenesis of Neurons in Human Sympathetic Ganglia - A Quantitative Study

Author(s): Kiran S.

Vol. 53, No. 2 (2004-07 - 2004-12)

Kamineni Institute of Medical Sciences, Narketpally, Nalgonda Dist., Andhra Pradesh.

Abstract:

To get a correct picture of the cell dynamics involved in the development of the human sympathetic neurons a quantitative study of the developing neurons becomes essential. A morphometric analysis, stereological and population studies were undertaken. For this study, a total of 102 samples were taken from superior cervical ganglia (SCG), thoracic ganglia (THG), and lumbar ganglia (LLG), of gestational ages ranging from 8th week to full term.

The morphometric study covered the cell dimensions and volumes of developing sympathetic neurons, dimensions and volumes of nuclei of the neurons. For this study a stage micrometer of 1mm. and an eyepiece micrometer was used. From the above results coefficients were drawn between cell and nuclear volume. Under the stereological study numerical density of the developing neurons of different gestational ages was assessed. For this study an eyepiece graticule with 6400 sq.mm area was used and stereological methods stipulated by Weibel were made use of. Under population study volume of ganglia and the total number of neurons per ganglion were analyzed. For this number of cells per mm3 x total volume of the ganglion formula was used. The results were compared with existing literature.

The morphometric studies reveal that the sympathoblasts are rounded in shape between 12th to 16th week of gestation. They change into oval shaped neurons by 18th week of gestation which continues throughout gestation. The neurons increase in volume by

15.7 times in SCG and 10 times in trunk ganglia by the time sympathoblasts reach the end of differentiation phase. During maturation phase they show a gradual increase of twice their size. The neuronal volume reaches 1228 M3 in SCG, 834 M3 in THG and 776 M3 in LLG by full term. The cells of the SCG differentiate earlier and they are one and a half times bigger cells compared with thoracic or lumbar ganglion neurons. Nucleus cell coefficient is 1 in sympathoblast stage, 1:4.2 in trunk ganglia and 1:5.2 in SCG at 24 weeks and reaches 1:5.2 to 1:5.6 proportion by full term. Stereoligcal studies reveal that at 12 weeks the sympathoblasts were around 25 to 26 lakh cells per cubic millimeter . They reduce to 66 to 74 thousand cells per cubic millimeter by 22-24 weeks. This is due to increase in cell size and the formation of satellite glial cells around this time. Beyond this stage though there is a reduction in number of cells per graticule that is more attributed to increase in cell size. In SCG the neurons are far bigger in comparison with THG and LLG.

An analysis of ganglionic volume shows that the size increases throughout gestation and the SCG is 11-12 times bigger compared to the trunk ganglia at full term. The population study shows that 15% sympathoblasts in SCG and 10% in trunk ganglia differentiate to form neurons. It also shows that neurons are added to ganglia throughout gestation. A comparative study of present work with existing literature shows that the neurons are added to the ganglia beyond birth and they increase in cell size.

Keywords: sympathetic ganglia, sympathetic neurons, quantitative study, stereological study, population study, dimensions, human fetus.

Introduction:

Sympathetic ganglia comprise of neurons, satellite cells, Schwann cells and connective tissue. Earlier Kiran (2002) studied the histogenesis of neurons of the sympathetic ganglia in humans. According to the author the histogenesis showed the following pattern. The sympathetic neurons were primitive migratory cells with cytoplasmic processes in 8th and 10th week ganglia. In 12th week ganglia they got rounded off and formed sympathoblasts. In 14th and 16th week ganglia the sympathoblasts showed aggregation into rosette formation. In 18th and 20th week ganglia, the cells in the rosette showed enlargement, and presented a heterochromatic nucleus. In 22nd and 24th week ganglia the cells showed euchromatic nucleus and axon formation. In 26th and 28th week ganglia the cells showed irregular appearance with the formation of dendrites, accumulation of cytoplasmic organelles and the nucleus showed a prominent nucleolus. In ganglia of 30th week to full term, the cells showed increase in size, increase in cytoplasmic organelles and formation of a capsule of satellite cells. The author described the development in four stages - (1) the primitive stage (8 to10 weeks), sympathoblast stage (12 to16 weeks), differentiation stage (18 to 28 weeks) and maturation stage (30 to 40 weeks).

To get the correct picture of the cell dynamics involved in the histogenesis of neurons of sympathetic ganglia quantitative, stereological and population studies become essential. Mayhew (1983) and Weibel (1969,1979) are the pioneers in applying the stereological principles to microanatomy. Pover and Cogestall (1991), Bolander et al (1991) and Abercombe (1946) applied these principles to understand the nervous system and revolutionalized this field. Though quantitative assessment of the adult sympathetic ganglia by de Castro (1932), Ebbeson (1968), Robinson and Gabella (1988) are available for some lower forms and humans, a thorough analysis of the developing sympathetic ganglia is still scarce. Hence the present study was undertaken on human fetuses.The present quantitative assessment is planned to study three aspects. (1) Morphometric study - this is to substantiate the growth patterns exhibited by the developing neurons, (2) The stereological study - is aimed at understanding the dispersion of the developing neurons at different gestational ages and (3) Population study - neurons exhibited different stages in their development. The population study was planned to get the exact numbers at different gestational ages.

The above quantitative studies were planned on samples of superior cervical ganglia, thoracic ganglia and lumbar ganglia and comparisons accomplished at definitive age groups. Same details are to be compared with the existing literature in this field. These are expected to throw light on the cell dynamics involved in the histogenesis of neurons of sympathetic ganglia.

Material and Methods:

17 human fetuses were obtained from the Department of Obstetrics and Gynaecology Department of Sri RamaChandra Medical College and Research Institute, Porur, Chennai, after obtaining necessary permission from concerned parents. These were well preserved fresh abortuses. On receiving the specimen, crown rump length measurements were made and gestational age was recorded. Langman (1981).

The youngest fetus obtained was of eight weeks of gestation. Beyond this one fetus for every two weeks of gestation, upto 36 weeks and two full term fetuses totaling 17 fetuses were received. Fetuses between 8th and 10th week were not considered for this study as they were at a primitive stage of development. For convenience of recording the quantitative details, fetuses were grouped into 8 groups - Group - I 12th week, Group - II 14th and 16th week, Group - III 18th and 20th week, Group - IV 22nd and 24th week, Group V 26th and 28th week, Group - VI 30th and 32nd week, Group VII 34th and 36th week and Group - VIII 2 full term fetuses. As the study was of the complete development of sympathetic chain, and since the area of supply of each ganglion is different it was decided to take 6 samples from each fetus, three from the right side (sample I) and three from left side (Sample II). They are superior cervical ganglion with a part of the chain labeled as SCG, thoracic chain with three ganglia and the interconnecting chain opposite thoracic vertebra 3rd to 6th labeled as THG, lower lumbar chain with three ganglia and interconnecting chain opposite to lumbar vertebrae 3rd to 5th labeled as LLG. These tissues were processed for paraffin blocking by routine histological procedure (Drury and Wallington 1980). A total of 102 blocks were used in this study.

From each fetus, one set (sample I) of SCG, THG and LLG was cut longitudinally and other set (sample II) of SCG, THG and LLG was cut transversely. 6mm serial sections were taken from each block. The slides were serially numbered with three sections on each slide (Sack 1963). While numbering 6 digits were used, the first two digits denote the age of fetus, the next two digits denote the position, the last two digit denotes the block number and beyond this number of slides was put with a - so that 260102-4 refers to the fourth slide (with three sections) of the 26th week old superior cervical ganglion's second block.

All even numbered sections were stained with hematoxylin and eosin to study the morphological features and general histological details. The odd numbered sections were used for special neurological stains. Following quantitative studies were undertaken on the H & E stained sections. For the morphometric and stereological study cells of all the eight groups were recorded and were analyzed according to their developmental stages. For population studies, except the first group, all the other seven groups from Group II to VIII that is from 16th week to full term only were considered, as ganglion formation could be noted only from sixteen weeks of gestation (Kiran 2002).

Morphometric Studies:

For this study longitudinally cut sections were used. A stage micrometer (1 mm scale to 100 divisions) and an eye piece micrometer with 100 divisions at 1,000 time magnification (oil immersion objective with 10x eyepiece) were used. At this magnification each eyepiece division corresponds to 1mm3, with this calibrated eye piece the length and breadth of the cells were recorded. The lengths of all the cells were added and their average was considered as average length. The breadths of all the cells were added and the average was considered as the average breadth. For this recording at random 10 cells per slide and 10 slides from each group were recorded. That is dimensions of 100 cells were recorded for each group. Volume of each cell was calculated by using formula ab2 /6 for oval structures. Volumes of individual cells were added and its average was considered as the average volume. Using the above formulae dimensions and volumes of sympathoblasts, nucleus of neurons and neurons were recorded for all groups. Comparative coefficients were drawn for nucleus and cell body of neurons.

Stereological Analysis:

Microtome sections describe the biological structures quantitatively in two dimensions. To understand these as multidimensional structures in space, stereological methods are made use of. This gives the estimate of mean values of the objects within structures and gives a descriptive infrastructure. This restores most of the information lost in sectioning Bolander et al (1991) and Weibel (1979). For this study transversely cut sections were used. An eye piece graticule with an area of 6400 mm2(80mm x 80 mm) under 1000 times magnification (oil immersion objective with 10X eye piece) was used for counting the cells in an area.

Table - I Cell Dimensions

Name Dimensions Group 1
(12)
weeks
GROUPS II
(14 to 16)
Weeks
GROUP III
(18 to 20)
Weeks
GROUP IV
(22 to 24)
Weeks
GROUP V
(26 to 28)
Weeks
GROUP VI
(30 to 32)
Weeks
GROUP VII
(34 to 36)
Weeks
GROUP VIII
Full Term
SCG Average Length (µ) 3.94 4.4 6.5 9.7 12.80 13.81 14.15 15.60
Average Breadth (µ) 3.49 4.05 5.7 7.5 9.36 10.02 10.25 11.70
Average Volume (µ3) 27.5 41.4 112.83 313.3 645.0 797.6 855 1228.2
THG Average Length (µ) 3.85 4.65 5.8 9.46 11.20 11.54 12.3 13.05
Average Breadth (µ) 3.38 4.00 5.3 6.86 8.35 9.13 9.55 10.60
Average Volume (µ3) 25.3 42.7 89.20 256.0 449.2 553.3 645.9 834.4
LLG Average Length (µ) 3.52 4.25 5.6 941 10.74 11.56 11.65 12.89
Average Breadth (µ) 3.02 3.8 5.2 6.84 7.79 8.33 9.6 10.23
Average Volume (µ3) 24.6 35.2 79.99 253.2 374.8 461.4 617.6 776.0

The length and breadth are in µ and the volume is in µ3

Table - II: Nuclear Dimensions

Name Dimensions Group 1
(12)
weeks
GROUPS II
(14 to 16)
Weeks
GROUP III
(18 to 20)
Weeks
GROUP IV
(22 to 24)
Weeks
GROUP V
(26 to 28)
Weeks
GROUP VI
(30 to 32)
Weeks
GROUP VII
(34 to 36)
Weeks
GROUP VIII
Full Term
SCG Average Length (µ) 3.94 4.4 5.5 5.10 6.80 6.86 7.05 7.82
Average Breadth (µ) 3.49 4.05 4.9 4.43 5.34 5.85 6.15 6.96
Average Volume (µ3) 27.5 41.4 49.9 57.5 113.1 143.6 153.3 217.9
THG Average Length (µ) 3.85 4.65 4.32 5.3 5.13 5.79 6.17 6.34
Average Breadth (µ) 3.38 4.00 4.6 4.8 5.85 5.85 6.65 6.69
Average Volume (µ3) 25.3 42.7 58.6 60.1 91.4 105.4 129.5 162.6
LLG Average Length (µ) 3.52 4.25 5.3 5.20 5.89 6.40 6.65 6.68
Average Breadth (µ) 3.02 3.8 4.5 4.8 5.16 5.49 5.87 6.05
Average Volume (µ3) 24.6 35.2 54.3 60.8 90.2 110.8 131.7 150.5

The length and breadth are in µ and the volume in µ3 m.

Table - III: Average Volume of Cell, Nucleus & Coefficient of Volumes

Name Dimensions Group 1
(12)
weeks
GROUPS II
(14 to 16)
Weeks
GROUP III
(18 to 20)
Weeks
GROUP IV
(22 to 24)
Weeks
GROUP V
(26 to 28)
Weeks
GROUP VI
(30 to 32)
Weeks
GROUP VII
(34 to 36)
Weeks
GROUP VIII
Full Term
SCG Cell (µ3) 27.5 41.4 75.9 313.3 645.0 797.6 855 1228.2
Nucleus (µ3) 27.5 41.4 49.9 57.5 113.1 143.6 153.3 217.9
Coefficient 1 1 1.34 5.4 5.7 5.5 5.5 5.6
THG Cell (µ3) 25.3 42.7 63.2 256.0 449.2 553.3 645.9 834.4
Nucleus (µ3) 25.3 42.7 58.6 60.1 91.4 105.4 129.5 162.6
Coefficient 1 1 1.08 4.2 4.9 5.2 5 5.2
LLG Cell (µ3) 24.6 35.2 61.7 253.2 374.8 461.4 617.6 776.0
Nucleus (µ3) 24.6 35.2 54.3 60.8 90.2 110.8 131.7 150.5
Coefficient 1 1 1.14 4.2 4.1 4.1 4.7 5.1

Table - IV: Numerical Densities of Neurons

Name   Group 1
(12)
weeks
GROUPS II
(14 to 16)
Weeks
GROUP III
(18 to 20)
Weeks
GROUP IV
(22 to 24)
Weeks
GROUP V
(26 to 28)
Weeks
GROUP VI
(30 to 32)
Weeks
GROUP VII
(34 to 36)
Weeks
GROUP VIII
Full Term
SCG Avg No/GRA 162.76 95.96 63.52 6.24 5.25 4.00 3.8 3.12
ND/mm3 26,17,700 14,66,300 8,86,100 66,700 48,000 34,800 32,600 24,800
THG Avg No/GRA 154.52 100 81.16 6.5 5.5 4.6 4.1 3.64
ND/mm3 25,12,300 15,13,300 11,63,400 71,700 54,400 43,900 37,800 31,900
LLG Avg No/GRA 149.56 110 82 6.75 6 5.25 4.8 3.9
ND/mm3 25,20,800 17,14,400 11,75,400 74,600 61,400 51,400 45,100 34,600

Avg No / GRA - Average Number per Graticute ND / mm3 - Numerical Density

Table - V : Population Studies

    GROUPS II
(14 to 16)
Weeks
GROUP III
(18 to 20)
Weeks
GROUP IV
(22 to 24)
Weeks
GROUP V
(26 to 28)
Weeks
GROUP VI
(30 to 32)
Weeks
GROUP VII
(34 to 36)
Weeks
GROUP VIII
Full Term
SCG Gang. Breadth & length in µ 684x3420 720x3600 756x3960 900x4860 1134x5760 1350x7020 1530x7560
volume in mm3 .82707638 .976043520 1.164982500 2.058941800 3.846635700 6.691235800 9.255637000
Population 12,12,630 8,64,833 77,638 98,784 1,33,840 2,81,126 2,29,524
THG Gang. Breadth & length in µ 360x1260 360x1440 432x1440 450x2124 630x2160 720x2160 810x2340
volume in mm3 0.85403808 0.096043520 0.174064860 0.224523900 0.448369990 0.585626110 0.802948300
Population 1,28,630 1,11,686 12,475 12,185 19,667 22,113 25,583
LLG Gang. Breadth & length in µ 324x900 360x1224 450x1440 504x2160 684x2052 720x1980 810x2160
volume in mm3 0.048199680 0.082692576 0.152506800 0.23535000 0.495762160 0.536823930 0.741183040
Population 1,20,998 96,382 11,339 14,429 25,443 24,173 25,638

ID / mm3 - Numerical density per millimeter cube.

Therein, the cells appearing in the focal plane of the microscope and falling within the graticular area were counted as per the optical dissector method of Gunderson and Jenson (1987) and Gunderson et al (1988). As the ganglia were too small up to 20th week of gestation 3 graticules per slide and 8 to 9 slides per group to a total of 25 graticules were counted. Beyond 20th week of gestation 5 graticules per slide and 10 slides of a group totaling 50 graticules were counted. The averages were calculated and were considered as the average number of cells per graticular area. From the above, numerical densities were calculated by using the formula ND=NA/D+T where ND is the number of cells per cubic millimeter, NA is mean number of cells per graticule, D is the mean diameter of the cell and T is the thickness of the slide Weibel (1979) and Sherif (1953). Average of both length and breadth put together was considered as mean diameter, and T was 6 µ.

Population Studies:

Neuronal number per ganglion at different gestational ages were studied in 21 samples, one each of SCG, THG and LLG of Group II to Group VIII. As the ganglia were spindle shaped, after going through all the sections carefully, the central longitudinal section was selected. Length and breadth of the ganglion was calculated by using stage and eyepiece micrometer from which the volume of the ganglion was calculated by using ab2 p/6 Physics formula. Total number of cells in a particular ganglion was calculated by using the formula - Number of cells /mm3 X total volume of the ganglion, Saleem and Krishnamurthy (1988). Vertical comparisons of the population were drawn for each gestational age group.

Results:

Morphometric Studies:

Results of morphometric study are presented in three tables. Table 1 shows the average length breadth and volume of 100 cells in each group.

Table II shows the average length, breadth and volume of nucleus of 100 cells in each group

Table III shows volumes of cells, volumes of nucleus of the cells and the cell nucleus proportion in all the groups.

Stereological Analysis:

Table IV shows the average number of cells per 25 graticules in Group I to III and 100 graticules between Group IV to VIII. It also shows the cell density per millimeter cube in all the ganglia

Population Studies:

Table V shows the ganglia sizes, volumes and the total number of cells in each ganglia in the age groups between 14 weeks to full term.

Discussion:

The morphological details of the developing cells in sympathetic ganglia were published by Kiran (2002). According to this report, the cells are in a primitive stage between 8th and 10th week. It is a migratory cell and shows cytoplasmic processes. Between 12th and 16th week of gestation (Group I and II) they are small rounded cells with a heterochromatic nucleus filling the whole cell and these are defined as sympathoblasts. Upto 28 weeks (Group III to V) they are differentiating neurons at which time they accumulate cytoplasmic organelles, put forth processes, the axon and dendrites. Upto full term (VI to VIII) they are maturing cells where they increase in cell size and become functional. The morphometric analysis was done keeping the above morphological details as yard sticks.

Morphometric Studies:

The present study of the cell, nuclear dimensions and volumes in all the ganglia show that at 16 weeks of gestation (Group II) the THG cells are bigger in size with 42.70 µ3, the SCG cells are next with 41.4 µ3 and LLG cells are the smallest with 35.2 µ3. It is a known fact that the sympathetic chain is initially laid in the trunk region, Romanes (1981) and Gabella (1976) and ascends upto cervical level and descends down to lumbar level. The present study gives an indication that the migration into the lumbar region is slightly delayed compared to cervical level from thoracic region. The present study shows that at this stage the cell, nuclear coefficient stands at 1. This shows that the nucleus fills the whole cell. Page (1986) identified rounded cells with scanty cytoplasm and nucleus filling the whole cell as sympathoblasts. Appenzeller(1920) and Kiran (2002) identified the sympathoblasts throughout the chain.

The sympathoblasts which are small and rounded cells differentiate and mature to form oval sympathetic neurons. The same was noted in new born, 10, 20 and 30 days old kittens by Masliukov (2001). Kiran (2002) noted that the sympathoblasts get committed to form neurons by 18th week and differentiate upto 28 weeks to form neurons. Present morphometric study shows that during this differentiation phase the length of the neurons is 20 to 28% more than the breadth of the neurons in all the ganglia of the sympathetic chain. This shows that the cells are more oval than rounded. The comparative analysis of the volume shows that the developing neurons have increased in size by 16 times in SCG from 41.4 µ3 to 645.00 µ3, 11 times in THG from 42.7 µ3 to 449.2 µ3 and 7.2 times in LLG from 35.2 µ3 to 374.8 µ3. The nucleus has increased in size by 3 times in SCG, 2.2 times in THG and 2.6 times in LLG. The cell nuclear coefficient shows that the nuclei are almost of the same size in all the ganglia but the increase in the cell size is due to increase in cytoplasm.

Fig 1

Missing Image

A transverse section through the Thoracic Ganglion of 14cm CRC (16 weeks) fetus.

Fig 2

Missing Image

A transverse section through the Thoracic Ganglion of 16 cm CRL (18 weeks) fetus, showing the oval appearance of the neurons.

Fig 3

Missing Image

A longitudinal section through the Lumbar Ganglion of 22 cm CRL (24 weeks) fetus, showing enlarged neurons with conical processes.

Fig 4.

Missing Image

A transverse section through the Superior Cervical Ganglion of 28 cm CRL (28 weeks) fetus, showing clear multipolar neurons with big open phase nucleus and a prominent nucleolus.

There is a second gradual increase in size of the cells during the maturation process (Group VI to VIII) by almost twice its size between 28 weeks to full term, from 645 mm3 to 1228.2 mm3 in SCG, 449.2 mm3 to 834.4 mm3 in THG, and from 374.8 mm3 to 776 mm3 in LLG. At full term the SCG cells are almost 1.5 times bigger than THG or LLG neurons. de Castro (1932) also recorded that the SCG cells are one and a half times bigger than the trunk ganglia in human adults. The nucleus of the neurons also has increased in size, but only twice in SCG and 1.6 times in THG and LLG. The cell nuclear coefficient shows that the cell is around 5 times bigger compared to its nuclear volume during this stage.

A close observation of the cell sizes and volumes of different gestational ages of SCG and THG and LLG shows that the cells of SCG differentiate earlier compared to the trunk sympathetic ganglia. An et al (2002) observed the same in developing zebra fish.

There is a gradual increase of cell volumes as well as nuclear volume to full term and the proportion is that the cell is 5.2 times bigger compared to its nucleus. A overall analysis shows that by 22 to 24 weeks the nuclear volume in comparison to the cell volume is achieved. This is mainly due to the state of the nucleus from heterochromatic nucleus of the sympathoblasts to the euchromatic nucleus of the developing neuron (Kiran 2002). The total increase of the cell volume and the proportionate increase of the nucleus kept pace with each other to around 5 times cell volume compared to the nuclear volume.

Stereological Study:

Stereological study of the sympathetic ganglia gives us the idea about the disposition of the cells within the ganglia. The neural crest cells migrate to a position dorsilateral to the aorta to form sympathetic chain Romanes (1981) and Langman (1981). They are multipotent cells and give rise to clones of neurons and glia Roufa et al (1996) and Anderson (1992). The present stereological study proves the above facts. In Group I when the undifferentiated cells transform into sympathoblasts162.76 cells in SCG, 154.52 cells in THG and 149.56 cells in LLG could be seen per graticular area. The numerical densities per cubic millimeter were 26,17,700 cells in SCG, 25,12,300 cells in THG and 25,20,800 cells in LLG at 16 weeks of gestation. This shows that the sympathoblasts are very closely packed.

In Group II the cells per graticule were reduced to 95.96 in SCG,100 in THG and 110in LLG. Number of cells per millimeter cube were reduced to 14,66,300 cells in SCG, 15,13,300 cells in THG and 17,14,400 in LLG. At this time the sympathoblasts were separated and arranged in a rosette formation (Kiran 2002). This could be the reason for the spread of the cells.

In Group III the cells per graticule were reduced to 63.52 in SCG, 81.16 in THG and 82 in LLG. Number of cells per millimeter cube were reduced to 8,86,100 cells in SCG, 11,63,400 cells in THG and 11,75,400 in LLG. At this time few of the cells in the rosettes showed enlargement(Kiran 2002).This could be the reason for the spread and less number of cells per graticule.

In Group IV the cells per graticule were reduced to 6.24 in SCG, 6.5 in THG and 6.75 in LLG. Number of cells per graticule was reduced to 66,700 cells in SCG, 71,700 cells in THG and 74,600 in LLG. At this time the cells enlarge in size, show the formation of axons. By this time the cells are easily identifiable as neurons. The remaining cells in the rosettes in due course form satellite cells Kiran and vatsala (1996). This could be the reason for the spread and less number of cells per graticule.

In Group V to Group VIII there is a gradual reduction in number of cells per graticular area as well as number of cells per cubic millimeter. The morphometric studies have shown a gradual increase in cell size around this time. The stereological study confirms that there are fewer cells per cubic millimeter area and are more spreadout with increasing gestational age.

Population Study:

An analysis of the total volume of the ganglion shows that the ganglionic volume has increased to 9.255 mm3 i.e. 11.5 times in SCG at full term in comparison to its size of .8270 mm3 at 16 weeks of gestation. Similarly the ganglionic volume has increased to .8029 mm3 i.e. 9.8 times at full term in THG, compared to .0854 mm3 at 16 weeks gestation. In LLG the ganglionic volume has increased to .7411 mm3 i.e. by 15 times by full term compared to its size of .0481 mm3 at 16 weeks of gestation. This shows that the LLG was relatively slower at the beginning of development.

Total population study shows only 77,638 cells out of 12,12,630 sympathoblasts showed differentiation in Group IV i.e. between 22 to 24 weeks of gestation in SCG that is one cell differentiated into neuron out of 15 sympathoblasts. Similarly in THG 12,475 cells out of 1,28,630 sympathoblasts differentiated into neurons. In LLG 11,339 cells differentiated into neurons out of 1,20,998 sympathoblasts. That is one neuron differentiated out of 10 sympathoblasts in trunk ganglia. The remaining cells in the rosettes formed satellite cells (Kiran and Vatsala 1996).

Total population of sympathetic neurons increased from 77,638 at 22 to 24 weeks to 2,29,524 at full term i.e. an increase of 3 times in SCG. In THG they have increased from 12,475 at 22 to 24 weeks to 25,583 at full term, and in LLG they increased from 11,339 at 22 to 24 weeks to 25,638 at full term. In both the cases it is an increase of twice the number of neurons at full term compared to 22 to 24 weeks. This shows that the neurons are continuously differentiating and are added throughout gestation. A thorough investigation of developing neurons in sympathetic chain in Zebra fish by An et al (2002) showed that neurons are added into the ganglion throughout gestation and at least upto 4 weeks of neonatal period.

A vertical comparison of the population of neurons at full term of SCG, THG and LLG shows that the THG and LLG have almost the same number of neurons, however the SCG have 8.9 times more number of neurons compared to the THG and LLG. This could be due to the fact that SCG is a bigger ganglion compared to the others.

Comparative Study:

Ebbeson's (1968) report on SCG is the only work available on human adult sympathetic ganglia in literature. For convenience of comparison, two SCG of full term were investigated in the present study. This was compared with Ebbesons (1968 a) work on adult SCG (Table VI).

Table - VI: Comparative Study - Superior Cervical Ganglion

No. Age Volume ND/mm3 Total no. of Neurons
1 Adult 133.3 mm3 7,100 9,47,300
2 Adult 173.5 mm3 4,500 7,73,100
3 Full term 9.25 mm3 24,800 2,29,524
4 Full term 9.88 mm3 24,800 2,45,073

The ganglionic volume recorded by Ebbeson (1968) in the adult was 133.3 mm3 and 173.5 mm3, whereas in SCG at full term it was recorded as 9.25 mm3 and 9.88mm3. This shows that an increase of 14 to 16 times in size from full term to adulthood.

Total number of cells per ganglion in adult SCG is 9,47,300 to 7,73,100. In the present study total number of cells ranges from 2,29,524 to 2,45,073. The packaging density of neurons per cubic mm in adult SCG was 7,100 and 4,500 and in comparison in our present study neurons per cubic mm was 24,800. Though Ebbeson's work showed a wide range in the volume and total population between the two ganglia recorded by him, the present work showed a variation within narrow limits. Though it is difficult to draw real conclusions under the above circumstances the following facts can still be ascertained. (1) Total number of cells increased from full term to adult stage shows that the cells are added beyond full term. (2) looking at the packaging density at full term and adult shows that the cell size has increased.

Summary:

Morphometric studies of sympathetic ganglia ranging from 8th week of gestation to full term reveal that the primitive migratory cells of 8th to 10th week settle down to form the rounded sympathoblasts by 12 to 16 weeks. At this stage, analysis of cell dimensions and volume shows that the LLG cells are smaller compared to SCG and THG. This could be due to the delayed migration into the lower lumbar region. The rounded sympathoblast change into an oval shaped neuron during differentiation phase of the development i.e. between 18 to 28 weeks of gestation. The neurons increase a great deal in size by 15.7 times in SCG, 10.5 times in THG and 10.6 times in LLG. During maturation phase of the neurons i.e. up to full term there is a gradual increase of cells to twice their size. The cells of the SCG differentiate earlier and they are one and a half time bigger cells compared to thoracic or lumbar ganglion neurons. Nucleus of the developing neurons has increased 2 to 2.7 times in size from sympathoblast cell stage to a differentiating cell stage at 18 weeks of gestation. Further it increases gradually in size in proportion to the cell size. In sympathoblasts the nucleus fills the cell. By 18 weeks the developing neurons show a marginal increase in size and they reach 5.4 times the nuclear size in SCG and 4.2 times in trunk ganglia by 22 to 24 weeks of gestation. The nuclear cell proportion lies at 1:52 in trunk ganglia and 1:5.6 in SCG at full term.

Stereological studies reveal that at 12 weeks the sympathoblasts were between 25 to 26 thousand cells per cubic millimeter. By 16 weeks they are 14 thousand cells in SCG to 15 thousand cells in THG to 17 thousand cells per cubic millimeter in LLG. A vertical comparison at this stage shows that the SCG cells are bigger in size compared to others and the lumbar cells are the smallest. At 22 to 24 weeks of gestation the cell number is very much reduced. This could be due to increase in cell size and the formation of satellite glial cells at this stage. Beyond this stage though there is a reduction in number of cells per graticule that is more attributed to increase in cell size. In SCG the neurons are far bigger in comparison with THG and LLG.

An analysis of the ganglionic volume of different gestational ages shows that there is a gradual increase in volumes of the ganglia with increasing age. Though the LLG is much smaller in size at 16 weeks of gestation they catch up with other trunk ganglia by full term. The SCG is 11 to12 times bigger in size compared to the trunk ganglia at full term. The total population study shows that only 15 sympathoblasts in SCG and 10 sympathoblasts in trunk ganglia differentiated to form neurons at 22 to 24 weeks gestation. It also shows that neurons are continuously added throughout gestation to the tune of thrice the number in SCG and twice the number in trunk ganglia. A comparative study of the present work with existing literature on adult sympathetic ganglia shows that the neurons are added into the ganglia beyond birth and they further increase in size.

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