- Email: [email protected]
Total neuronal numbers of rat lumbosacral primary afferent neurons do not change with age
Neuroscience Letters 304 (2001) 149±152
www.elsevier.com/locate/neulet
Total neuronal numbers of rat lumbosacral primary afferent neurons do not change with age H.A. Mohammed, R.M. Santer* School of Biosciences, University of Wales, Cardiff, P.O. Box 911, Cardiff CF10 3US, UK Received 26 February 2001; received in revised form 22 March 2001; accepted 23 March 2001
Abstract Total cell numbers and neuronal diameters of L6 and S1 dorsal root ganglia, which provide a sensory innervation to pelvic viscera were determined in young adult (3-months-old) and compared to those in old (24-months-old) male rats. Two methods of cell counting, serial (section) reconstructions and total pro®le counting, were used in this study. Our data showed that the total number of L6 and S1 dorsal root ganglia (DRG) cells and their diameters remain essentially constant from 3 to 24 months of age. These results have shown that rat DRG cell numbers do not change during adult life and that neurogenesis of DRG cells in adult rats or neuronal cell death in aged rats cannot be supported. These ®ndings are also consistent with other data supporting the maintenance of pelvic sensory innervation in old age. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dorsal root ganglia; Sensory neurons; Pelvic innervation; Serial reconstruction; Ageing; Rat
One of the most fundamental issues in current biology is how to maintain the critical balance between cell survival and death, both during adulthood and in ageing. For much of its history, the study of naturally occurring cell death in dorsal root ganglia (DRG) relied on inferences based on estimates of total neuron numbers in avian or amphibian species [1]. Maturation and ageing of nerve tissue is characterized by alterations in size and numbers of its structural components [18]. Since DRGs are part of the peripheral nervous system, their actual total number and diameter are of particular interest in the study of disorders involving peripheral nerves. DRG neurons are markedly diverse, and a number of classi®cation schemes have been used to divide the DRG neurons into subgroups based on size (large or small) and cytological (depending on cytoplasmic appearance), chemical or physiological properties [2]. Studies in sensory neuron numbers have showed different changes with age. Increases in cell numbers in the rat L4 and L5 DRG [5,6,16] as well as no changes in DRGs numbers [10,11] have been reported with age. Counting methods for histologically sectioned material fall into four categories: (1) pro®le counts (a pro®le is what is seen of a cell in a histological section); (2) serial (section) reconstruc* Corresponding author. Tel.: 144-29-20874842; fax: 144-2920875964. E-mail address: [email protected] (R.M. Santer).
tions; (3) assumption-based methods, which are pro®le counts plus geometric assumption that convert the pro®le counts into cell or synapse numbers; and (4) stereological methods [4]. Since the serial section reconstruction method provides an unbiased estimate of the total number of neurons in DRG [4,13], this method was used in the present study. To our knowledge, this method of cell counting has not been used in young and aged rats so far. Total pro®le counting method was also used as a comparison with serial reconstruction method. Therefore, the primary aim of the present study (as part of a wider investigation into the effects of age on the pelvic sensory innervation) was to elucidate whether there is any decrease or increase with age in the number and diameter of L6 and S1 DRG neurons. Five pairs of male Wistar rats aged 3 and 24 months were used in this study. Animals were perfused transcardially with a solution containing 4% paraformaldehyde in 100 mM phosphate-buffered saline (PBS) under deep anaesthesia with Euthatal (20 mg/ml pentobarbitone sodium; RhoÃne MeÂrieux, Dublin). The dorsal root ganglia at spinal levels L6 and S1 were removed and post®xed in 4% paraformaldehyde for 2 h. After buffer-washing, the ganglia were stored in PBS containing 30% sucrose prior to crysectioning at 8 mm. The sections were stained with aqueous 1% toluidine blue for 30 s, dehydrated in ascending series of alcohols and mounted in DPX (Fisher Scienti®c, UK). The neurones were characterized as A- or B-cells according to
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 78 1- 5
150
H.A. Mohammed, R.M. Santer / Neuroscience Letters 304 (2001) 149±152
the criteria given by Lieberman [14] and Harper and Lawson [8] in which DRG cells subdivided into two types of neurons, A-cells (large light) and B-cells (small dark) neurons. The large light cell population give rise to the fast-conducting myelinated Aa and Ab mechanoreceptive ®bres, while small dark neurons comprises mostly DRG neurons with Ad and C ®bres which convey nociceptive
signals [7,12,14,17,19,21]. In addition to the serial (section) reconstructions, other commonly used methods of pro®le counting [5,8,9,20,22] were employed also (i.e. counting of cell pro®les every third or sixth section, then multiplying the number of cells by three or six, respectively). In the serial reconstruction method, a complete series of sections were collected from each young and aged DRG. Each
Fig. 1. A light micrograph of two consecutive 8 mm sections through L6 DRG of young (A,B) and aged (C,D) rats. Note the large and small DRG cells with prominent nuclei. Most of these nuclei contain nucleolar pro®les, which have been numbered, followed from section to section and counted to yield the total numbers of cells. Scale bar (A,B) 25 mm; (C,D) 50 mm.
H.A. Mohammed, R.M. Santer / Neuroscience Letters 304 (2001) 149±152
151
Table 1 Number of neurons in L6 and S1 DRG of young (3-months-old) and aged (24-months-old) male rats using different methods a Methods
Young
Serial Recon. third section sixth section Mean ^ S.D a
Aged
L6
S1
L6
S1
16655 16180 15866 16230 ^ 397*
15315 14790 14320 14810 ^ 498*
15996 15644 15388 15680 ^ 305*
14887 14316 13912 14370 ^ 490*
P . 0:05.
section was digitally imaged on Leica DMR microscope ®tted with a Spot digital camera (Diagnostic instruments, Inc. USA) and analyzed with Image-Pro Plus version 4.0 (Media Cybernetics, USA). Each section was captured and saved in the computer. Only neuronal pro®les with a distinct nucleolus were numbered and counted, and if the following sections showed the same cell with its nucleolus, we only counted the cell with larger and more central nucleolus. Each section was then compared to the previous image in order to avoid double counting of neurons, and all cells were followed in the same way until their full extent was delineated. It was possible to compare two to three images at one time on a 19-inch monitor screen. This method allowed the cellular pro®les to be followed from section to section (Fig. 1), ensuring that each cell had been counted only once (serial reconstruction method) [3,4,13]. The counts from each section were then added together to ®nd the total number per ganglion. Then, these numbers were compared with those obtained by total pro®le counting method (every third or sixth section) and the difference in numbers between these methods were given as a percentage. In addition, 100 A-cells and 100 B-cells, from each young and aged DRG, were used for diameter and area measurements. For each age group, the means ^ SD of total cell numbers and diameters of L6 and S1 DRG neurons were analyzed and the results from both age groups were tested statistically using unpaired (two-tailed) Student's t-test and analysis of variance (ANOVA) and the differences of P , 0:05 were considered signi®cant. Serial reconstruction method, in comparison to other two methods, showed the highest number of neurons at all levels of DRGs examined in both age groups (Table 1). Counting of every third section method revealed a decrease in cell Table 2 Mean cell diameter of A- and B-cells in young and aged male rat L6 & S1 DRGs a A-cell* (mean ^ S.D)
L6 S1 a
B-cell* (mean ^ S.D)
Young
Aged
Young
Aged
36.4 ^ 8.4 35.8 ^ 8.1
37.3 ^ 7.8 35.5 ^ 7
20.6 ^ 2.5 20.4 ^ 2.4
20.4 ^ 2.5 20.3 ^ 2.8
P . 0:05.
numbers of 2.2±3.8%, while counting every sixth section produced a reduction of 3.8±6.5%, compared to the serial reconstruction method. The total number of cells obtained by the three methods of cell counting showed that L6 dorsal root ganglia had the highest numbers of neurons in both young and aged rats. Statistical analysis of mean cell numbers in young rat L6-S1 DRGs showed no signi®cant (P . 0:05) difference with those in aged rats. On the other hand, the mean cell diameters of A-cells in L6 DRG showed larger mean diameters, in both young (36.4 ^ 8.4 mm) and aged (37.3 ^ 7.8 mm) rats, than those in S1 DRG which demonstrated a mean cell diameter of 35.8 ^ 8.1 mm in young and 35.5 ^ 7 mm in aged rats (Table 2). In contrast, the diameters of young B-cells ranged from 20.4 mm in S1 DRG to 20.6mm in L6 DRG, while aged B-cell diameters varied between 20.3 mm in S1 DRG and 20.4 mm in L6 DRG. Mean cell diameters of both A- and B-cells in L6S1 DRGs showed no signi®cant differences (P . 0:05) with age. There are disagreements about numbers of DRG cells in the rat. Schmalbruch [20] pointed out that the counts of DRG cells in rat L3±L6 ganglia differ by as much as three-fold in different studies and that no study agrees with others. We believe that a major dif®culty in this regard is that different counting methods lead to different estimates of cell numbers. Therefore, one of the reasons for carrying out this study was to establish whether there is a difference in DRG cell numbers between young and aged rats and also to see if there is a real difference in estimating DRG cell numbers using serial (section) reconstructions and pro®le counting methods. The main ®ndings of the present study are that the numbers and diameters of L6 and S1 DRGs cells have remained essentially constant from 3 to 24 months of age. Further results have also shown the difference in cell numbers obtained by total pro®le counting at constant intervals (3 and 6 months) have decreased as the space intervals between the examined sections have increased from 3 to 6 months. These observations support the idea that serial (section) reconstructions method is more accurate for estimating the number of DRG cells and the counting pro®les every third or sixth sections can lead to a biased numbers. The results of the present study are in agreement with the ®ndings reported previously [11] where L4±L6 DRG cell numbers did not change signi®cantly from 3 to 22 months of
152
H.A. Mohammed, R.M. Santer / Neuroscience Letters 304 (2001) 149±152
age (although the total numbers of DRGs were below our ®gures). Moreover, the ®ndings of this study are in disagreement with those obtained by [5,16] in the rat L3±L6 DRG, where an increase in cell numbers (neurogenesis) was reported. These authors used different methods of counting: physical dissector and pro®le counting (one or more nucleoli within a nucleus). The heterogeneous distribution of neurons in the ganglia can lead to sampling errors of up to 50% and counting of nuclear/nucleolar pro®les can introduce a bias that can change the actual numbers of neurons [4]. In serial (section) reconstructions, on the other hand, there is no sampling or estimating; all the cells are reconstructed and the numbers of cells become known. Evidence in favour of neurogenesis would be the demonstration of cell division, either by mitotic ®gures or by tritiated thymidine autoradiography [11], and the demonstration of increases in sensory axon numbers either in the dorsal roots or in peripheral nerves. Mitotic ®gures have not been reported in adult mammalian DRG cells [11] nor were any seen in this investigation and with regard to the numbers of axons, no postnatal increase in myelinated axon numbers has been observed in dorsal roots [5]. In addition, the numbers of both myelinated and unmyelinated axons showed no changes during adulthood [10]. Thus, in the rat DRG the idea of neurogenesis cannot be supported. The data presented above is consistent with a recent study [15] in which no signi®cant changes in the sensory neuropeptidergic innervation of pelvic viscera have been reported. The authors would like to acknowledge the Ministry of Higher Education, Saudi Arabia and the King Abdulaziz University, Jeddah for the scholarship during which this work was undertaken. [1] Berg, S.J. and Farel, P.B., Developmental regulation of sensory neuron number and limb innervation in the mouse, Dev. Brain Res., 125 (2000) 21±30. [2] Bergman, E., Carlsson, K., Liljeborg, A., Manders, E., Hokfelt, T. and Ulfhake, B., Neuropeptides, nitric oxide synthase and GAP-43 in B4-binding and RT97 immunoreactive primary sensory neurons: normal distribution pattern and changes after peripheral nerve transection and aging, Brain Res., 832 (1999) 63±83. [3] Coggeshall, R.E., A consideration of neural counting methods, Trends Neurosci., 15 (1992) 9±14. [4] Coggeshall, R.E. and Lekan, H., Methods for determining numbers of cells and synapses: a case for more uniform standards of review, J. Comp. Neurol., 364 (1996) 6±15. [5] Devor, M., Govrin-Lipmann, R., Frank, I. and Raber, P., Proliferation of primary sensory neurons in adult rat dorsal root ganglion and the kinetics of retrograde cell loss after
[6] [7]
[8] [9]
[10] [11] [12]
[13] [14] [15] [16]
[17] [18] [19]
[20] [21] [22]
sciatic nerve section, Somatosensory Res., 3 (1985) 139± 167. Devor, M. and Govrin-Lipmann, R., Neurogenesis in adult rat dorsal root ganglia: on counting and the count, Somatosensory Motor Res., 8 (1991) 9±12. Duce, I.R. and Keen, P., An ultrstructural classi®cation of the neuronal cell bodies of the rat dorsal root ganglion using zinc iodide-osmium impregnation, Cell Tiss. Res., 185 (1977) 263±277. Harper, A.A. and Lawson, S.N., Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurones, J. Physiol., 359 (1985) 31±46. Himes, B.T. and Tessler, A., Death of some dorsal root ganglion neurons and plasticity of others following sciatic nerve section in adult and neonatal rats, J. Comp. Neurol., 284 (1989) 215±230. Hulsebosch, C.L., Coggeshall, R.E. and Chung, K., Numbers of dorsal root axons and ganglion cells during postnatal development, Dev. Brain Res., 26 (1986) 105±113. La Forte, R.A., Melville, S., Chung, K. and Coggeshall, R.E., Absence of neurogenesis of adult rat dorsal root ganglion cells, Somatosensory Motor Res., 8 (1991) 3±7. Lawson, S.N., the postnatal development of large light and small dark neurons in mouse dorsal root ganglia: a statistical analysis of cell numbers and size, J. Neurocytol., (1979) 275. Ledda, M., Barni, L., Altieri, L. and Pannese, E., Decrease in the nucleo-cytoplasmic volume ratio of rabbit spinal ganglion neurons with age, Neurosci. Lett., 286 (2000) 171±174. Lieberman, A.R., The peripheral nerve, In D.N. Landon (Ed.), Sensory Ganglia, Chapman and Hall, London, 1976, pp. 188±278. Mohammed, H. and Santer, R.M., Sensory peptidergic innervation of the lower urinary tract in young adult and aged rats, J. Anat., 196 (2000) 136. Popken, G.J. and Farel, P.B., Sensory neuron number in neonatal and adult rats estimated by means of stereologic and pro®le-based methods, J. Comp. Neurol., 386 (1997) 8± 15. Rambourg, A., Clermont, Y. and Beaudet, A., Ultrstructural features of six types of neurons in rat dorsal root ganglia, J. Neurocytol., 12 (1983) 47±66. Rao, R. and Krinkle, G., Changes with age in the number and size of myelinated axons in the rat L4 dorsal spinal root, Acta Anat., 117 (1983) 187±192. Sommer, E.W., Kazimierczak, J. and Droz, B., Neuronal subpopulations in the dorsal root ganglion of the mouse as characterized by combination of ultrastructural and cytochemical features, Brain Res., 346 (1985) 310±326. Schmalbruch, H., The number of neurons in dorsal root ganglion L4±L6 of the rat, Anat. Rec., 219 (1987) 315±322. Tandrup, T. and Brendgaard, H., Number and volume of rat dorsal root ganglion cells in acrylamide intoxication, J. Neurocytol., 23 (1994) 242±248. Tandrup, T., Are the neurons in the dorsal root ganglion pseudounipolar? A comparison of the number of neurons and number of myelinated and unmyelinated ®bres in the dorsal root, J. Comp. Neurol., 357 (1995) 341±347.
www.elsevier.com/locate/neulet
Total neuronal numbers of rat lumbosacral primary afferent neurons do not change with age H.A. Mohammed, R.M. Santer* School of Biosciences, University of Wales, Cardiff, P.O. Box 911, Cardiff CF10 3US, UK Received 26 February 2001; received in revised form 22 March 2001; accepted 23 March 2001
Abstract Total cell numbers and neuronal diameters of L6 and S1 dorsal root ganglia, which provide a sensory innervation to pelvic viscera were determined in young adult (3-months-old) and compared to those in old (24-months-old) male rats. Two methods of cell counting, serial (section) reconstructions and total pro®le counting, were used in this study. Our data showed that the total number of L6 and S1 dorsal root ganglia (DRG) cells and their diameters remain essentially constant from 3 to 24 months of age. These results have shown that rat DRG cell numbers do not change during adult life and that neurogenesis of DRG cells in adult rats or neuronal cell death in aged rats cannot be supported. These ®ndings are also consistent with other data supporting the maintenance of pelvic sensory innervation in old age. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dorsal root ganglia; Sensory neurons; Pelvic innervation; Serial reconstruction; Ageing; Rat
One of the most fundamental issues in current biology is how to maintain the critical balance between cell survival and death, both during adulthood and in ageing. For much of its history, the study of naturally occurring cell death in dorsal root ganglia (DRG) relied on inferences based on estimates of total neuron numbers in avian or amphibian species [1]. Maturation and ageing of nerve tissue is characterized by alterations in size and numbers of its structural components [18]. Since DRGs are part of the peripheral nervous system, their actual total number and diameter are of particular interest in the study of disorders involving peripheral nerves. DRG neurons are markedly diverse, and a number of classi®cation schemes have been used to divide the DRG neurons into subgroups based on size (large or small) and cytological (depending on cytoplasmic appearance), chemical or physiological properties [2]. Studies in sensory neuron numbers have showed different changes with age. Increases in cell numbers in the rat L4 and L5 DRG [5,6,16] as well as no changes in DRGs numbers [10,11] have been reported with age. Counting methods for histologically sectioned material fall into four categories: (1) pro®le counts (a pro®le is what is seen of a cell in a histological section); (2) serial (section) reconstruc* Corresponding author. Tel.: 144-29-20874842; fax: 144-2920875964. E-mail address: [email protected] (R.M. Santer).
tions; (3) assumption-based methods, which are pro®le counts plus geometric assumption that convert the pro®le counts into cell or synapse numbers; and (4) stereological methods [4]. Since the serial section reconstruction method provides an unbiased estimate of the total number of neurons in DRG [4,13], this method was used in the present study. To our knowledge, this method of cell counting has not been used in young and aged rats so far. Total pro®le counting method was also used as a comparison with serial reconstruction method. Therefore, the primary aim of the present study (as part of a wider investigation into the effects of age on the pelvic sensory innervation) was to elucidate whether there is any decrease or increase with age in the number and diameter of L6 and S1 DRG neurons. Five pairs of male Wistar rats aged 3 and 24 months were used in this study. Animals were perfused transcardially with a solution containing 4% paraformaldehyde in 100 mM phosphate-buffered saline (PBS) under deep anaesthesia with Euthatal (20 mg/ml pentobarbitone sodium; RhoÃne MeÂrieux, Dublin). The dorsal root ganglia at spinal levels L6 and S1 were removed and post®xed in 4% paraformaldehyde for 2 h. After buffer-washing, the ganglia were stored in PBS containing 30% sucrose prior to crysectioning at 8 mm. The sections were stained with aqueous 1% toluidine blue for 30 s, dehydrated in ascending series of alcohols and mounted in DPX (Fisher Scienti®c, UK). The neurones were characterized as A- or B-cells according to
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 78 1- 5
150
H.A. Mohammed, R.M. Santer / Neuroscience Letters 304 (2001) 149±152
the criteria given by Lieberman [14] and Harper and Lawson [8] in which DRG cells subdivided into two types of neurons, A-cells (large light) and B-cells (small dark) neurons. The large light cell population give rise to the fast-conducting myelinated Aa and Ab mechanoreceptive ®bres, while small dark neurons comprises mostly DRG neurons with Ad and C ®bres which convey nociceptive
signals [7,12,14,17,19,21]. In addition to the serial (section) reconstructions, other commonly used methods of pro®le counting [5,8,9,20,22] were employed also (i.e. counting of cell pro®les every third or sixth section, then multiplying the number of cells by three or six, respectively). In the serial reconstruction method, a complete series of sections were collected from each young and aged DRG. Each
Fig. 1. A light micrograph of two consecutive 8 mm sections through L6 DRG of young (A,B) and aged (C,D) rats. Note the large and small DRG cells with prominent nuclei. Most of these nuclei contain nucleolar pro®les, which have been numbered, followed from section to section and counted to yield the total numbers of cells. Scale bar (A,B) 25 mm; (C,D) 50 mm.
H.A. Mohammed, R.M. Santer / Neuroscience Letters 304 (2001) 149±152
151
Table 1 Number of neurons in L6 and S1 DRG of young (3-months-old) and aged (24-months-old) male rats using different methods a Methods
Young
Serial Recon. third section sixth section Mean ^ S.D a
Aged
L6
S1
L6
S1
16655 16180 15866 16230 ^ 397*
15315 14790 14320 14810 ^ 498*
15996 15644 15388 15680 ^ 305*
14887 14316 13912 14370 ^ 490*
P . 0:05.
section was digitally imaged on Leica DMR microscope ®tted with a Spot digital camera (Diagnostic instruments, Inc. USA) and analyzed with Image-Pro Plus version 4.0 (Media Cybernetics, USA). Each section was captured and saved in the computer. Only neuronal pro®les with a distinct nucleolus were numbered and counted, and if the following sections showed the same cell with its nucleolus, we only counted the cell with larger and more central nucleolus. Each section was then compared to the previous image in order to avoid double counting of neurons, and all cells were followed in the same way until their full extent was delineated. It was possible to compare two to three images at one time on a 19-inch monitor screen. This method allowed the cellular pro®les to be followed from section to section (Fig. 1), ensuring that each cell had been counted only once (serial reconstruction method) [3,4,13]. The counts from each section were then added together to ®nd the total number per ganglion. Then, these numbers were compared with those obtained by total pro®le counting method (every third or sixth section) and the difference in numbers between these methods were given as a percentage. In addition, 100 A-cells and 100 B-cells, from each young and aged DRG, were used for diameter and area measurements. For each age group, the means ^ SD of total cell numbers and diameters of L6 and S1 DRG neurons were analyzed and the results from both age groups were tested statistically using unpaired (two-tailed) Student's t-test and analysis of variance (ANOVA) and the differences of P , 0:05 were considered signi®cant. Serial reconstruction method, in comparison to other two methods, showed the highest number of neurons at all levels of DRGs examined in both age groups (Table 1). Counting of every third section method revealed a decrease in cell Table 2 Mean cell diameter of A- and B-cells in young and aged male rat L6 & S1 DRGs a A-cell* (mean ^ S.D)
L6 S1 a
B-cell* (mean ^ S.D)
Young
Aged
Young
Aged
36.4 ^ 8.4 35.8 ^ 8.1
37.3 ^ 7.8 35.5 ^ 7
20.6 ^ 2.5 20.4 ^ 2.4
20.4 ^ 2.5 20.3 ^ 2.8
P . 0:05.
numbers of 2.2±3.8%, while counting every sixth section produced a reduction of 3.8±6.5%, compared to the serial reconstruction method. The total number of cells obtained by the three methods of cell counting showed that L6 dorsal root ganglia had the highest numbers of neurons in both young and aged rats. Statistical analysis of mean cell numbers in young rat L6-S1 DRGs showed no signi®cant (P . 0:05) difference with those in aged rats. On the other hand, the mean cell diameters of A-cells in L6 DRG showed larger mean diameters, in both young (36.4 ^ 8.4 mm) and aged (37.3 ^ 7.8 mm) rats, than those in S1 DRG which demonstrated a mean cell diameter of 35.8 ^ 8.1 mm in young and 35.5 ^ 7 mm in aged rats (Table 2). In contrast, the diameters of young B-cells ranged from 20.4 mm in S1 DRG to 20.6mm in L6 DRG, while aged B-cell diameters varied between 20.3 mm in S1 DRG and 20.4 mm in L6 DRG. Mean cell diameters of both A- and B-cells in L6S1 DRGs showed no signi®cant differences (P . 0:05) with age. There are disagreements about numbers of DRG cells in the rat. Schmalbruch [20] pointed out that the counts of DRG cells in rat L3±L6 ganglia differ by as much as three-fold in different studies and that no study agrees with others. We believe that a major dif®culty in this regard is that different counting methods lead to different estimates of cell numbers. Therefore, one of the reasons for carrying out this study was to establish whether there is a difference in DRG cell numbers between young and aged rats and also to see if there is a real difference in estimating DRG cell numbers using serial (section) reconstructions and pro®le counting methods. The main ®ndings of the present study are that the numbers and diameters of L6 and S1 DRGs cells have remained essentially constant from 3 to 24 months of age. Further results have also shown the difference in cell numbers obtained by total pro®le counting at constant intervals (3 and 6 months) have decreased as the space intervals between the examined sections have increased from 3 to 6 months. These observations support the idea that serial (section) reconstructions method is more accurate for estimating the number of DRG cells and the counting pro®les every third or sixth sections can lead to a biased numbers. The results of the present study are in agreement with the ®ndings reported previously [11] where L4±L6 DRG cell numbers did not change signi®cantly from 3 to 22 months of
152
H.A. Mohammed, R.M. Santer / Neuroscience Letters 304 (2001) 149±152
age (although the total numbers of DRGs were below our ®gures). Moreover, the ®ndings of this study are in disagreement with those obtained by [5,16] in the rat L3±L6 DRG, where an increase in cell numbers (neurogenesis) was reported. These authors used different methods of counting: physical dissector and pro®le counting (one or more nucleoli within a nucleus). The heterogeneous distribution of neurons in the ganglia can lead to sampling errors of up to 50% and counting of nuclear/nucleolar pro®les can introduce a bias that can change the actual numbers of neurons [4]. In serial (section) reconstructions, on the other hand, there is no sampling or estimating; all the cells are reconstructed and the numbers of cells become known. Evidence in favour of neurogenesis would be the demonstration of cell division, either by mitotic ®gures or by tritiated thymidine autoradiography [11], and the demonstration of increases in sensory axon numbers either in the dorsal roots or in peripheral nerves. Mitotic ®gures have not been reported in adult mammalian DRG cells [11] nor were any seen in this investigation and with regard to the numbers of axons, no postnatal increase in myelinated axon numbers has been observed in dorsal roots [5]. In addition, the numbers of both myelinated and unmyelinated axons showed no changes during adulthood [10]. Thus, in the rat DRG the idea of neurogenesis cannot be supported. The data presented above is consistent with a recent study [15] in which no signi®cant changes in the sensory neuropeptidergic innervation of pelvic viscera have been reported. The authors would like to acknowledge the Ministry of Higher Education, Saudi Arabia and the King Abdulaziz University, Jeddah for the scholarship during which this work was undertaken. [1] Berg, S.J. and Farel, P.B., Developmental regulation of sensory neuron number and limb innervation in the mouse, Dev. Brain Res., 125 (2000) 21±30. [2] Bergman, E., Carlsson, K., Liljeborg, A., Manders, E., Hokfelt, T. and Ulfhake, B., Neuropeptides, nitric oxide synthase and GAP-43 in B4-binding and RT97 immunoreactive primary sensory neurons: normal distribution pattern and changes after peripheral nerve transection and aging, Brain Res., 832 (1999) 63±83. [3] Coggeshall, R.E., A consideration of neural counting methods, Trends Neurosci., 15 (1992) 9±14. [4] Coggeshall, R.E. and Lekan, H., Methods for determining numbers of cells and synapses: a case for more uniform standards of review, J. Comp. Neurol., 364 (1996) 6±15. [5] Devor, M., Govrin-Lipmann, R., Frank, I. and Raber, P., Proliferation of primary sensory neurons in adult rat dorsal root ganglion and the kinetics of retrograde cell loss after
[6] [7]
[8] [9]
[10] [11] [12]
[13] [14] [15] [16]
[17] [18] [19]
[20] [21] [22]
sciatic nerve section, Somatosensory Res., 3 (1985) 139± 167. Devor, M. and Govrin-Lipmann, R., Neurogenesis in adult rat dorsal root ganglia: on counting and the count, Somatosensory Motor Res., 8 (1991) 9±12. Duce, I.R. and Keen, P., An ultrstructural classi®cation of the neuronal cell bodies of the rat dorsal root ganglion using zinc iodide-osmium impregnation, Cell Tiss. Res., 185 (1977) 263±277. Harper, A.A. and Lawson, S.N., Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurones, J. Physiol., 359 (1985) 31±46. Himes, B.T. and Tessler, A., Death of some dorsal root ganglion neurons and plasticity of others following sciatic nerve section in adult and neonatal rats, J. Comp. Neurol., 284 (1989) 215±230. Hulsebosch, C.L., Coggeshall, R.E. and Chung, K., Numbers of dorsal root axons and ganglion cells during postnatal development, Dev. Brain Res., 26 (1986) 105±113. La Forte, R.A., Melville, S., Chung, K. and Coggeshall, R.E., Absence of neurogenesis of adult rat dorsal root ganglion cells, Somatosensory Motor Res., 8 (1991) 3±7. Lawson, S.N., the postnatal development of large light and small dark neurons in mouse dorsal root ganglia: a statistical analysis of cell numbers and size, J. Neurocytol., (1979) 275. Ledda, M., Barni, L., Altieri, L. and Pannese, E., Decrease in the nucleo-cytoplasmic volume ratio of rabbit spinal ganglion neurons with age, Neurosci. Lett., 286 (2000) 171±174. Lieberman, A.R., The peripheral nerve, In D.N. Landon (Ed.), Sensory Ganglia, Chapman and Hall, London, 1976, pp. 188±278. Mohammed, H. and Santer, R.M., Sensory peptidergic innervation of the lower urinary tract in young adult and aged rats, J. Anat., 196 (2000) 136. Popken, G.J. and Farel, P.B., Sensory neuron number in neonatal and adult rats estimated by means of stereologic and pro®le-based methods, J. Comp. Neurol., 386 (1997) 8± 15. Rambourg, A., Clermont, Y. and Beaudet, A., Ultrstructural features of six types of neurons in rat dorsal root ganglia, J. Neurocytol., 12 (1983) 47±66. Rao, R. and Krinkle, G., Changes with age in the number and size of myelinated axons in the rat L4 dorsal spinal root, Acta Anat., 117 (1983) 187±192. Sommer, E.W., Kazimierczak, J. and Droz, B., Neuronal subpopulations in the dorsal root ganglion of the mouse as characterized by combination of ultrastructural and cytochemical features, Brain Res., 346 (1985) 310±326. Schmalbruch, H., The number of neurons in dorsal root ganglion L4±L6 of the rat, Anat. Rec., 219 (1987) 315±322. Tandrup, T. and Brendgaard, H., Number and volume of rat dorsal root ganglion cells in acrylamide intoxication, J. Neurocytol., 23 (1994) 242±248. Tandrup, T., Are the neurons in the dorsal root ganglion pseudounipolar? A comparison of the number of neurons and number of myelinated and unmyelinated ®bres in the dorsal root, J. Comp. Neurol., 357 (1995) 341±347.