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The regional distribution of nitric oxide synthase activity in the spinal cord of the dog
Brain Research Bulletin, Vol. 58, No. 2, pp. 173–178, 2002 Copyright © 2002 Elsevier Science Inc. All rights reserved. 0361-9230/02/$–see front matter
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The regional distribution of nitric oxide synthase activity in the spinal cord of the dog 1∗ Dáša C´ ˇ ıžková,1 Martin Maršala,2 Imrich Lukácˇ 3 and Jozef Maršala1 ˇ Nadežda Lukácová, 1 Institute
of Neurobiology, Slovak Academy of Sciences, Košice, Slovak Republic; 2 Anesthesiology Research Laboratory, 0818 University of California, La Jolla, CA, USA; and 3 Department of Neurosurgery, Medical Faculty of P. J. Šafárik University, Košice, Slovak Republic [Received 7 January 2002; Accepted 12 February 2002] been developed and it was clearly demonstrated that this staining is almost identical to histochemical staining for NADPHd. Therefore, both these different techniques are currently used to localize the neurons that are small in number and considered to be not more that 1–2% of total neuronal population [6]. However, the number of NADPHd-exhibiting and NOS immunoreactive neurons was found to be regionally different [39,40]. Moreover, biochemical analysis of cNOS activity documented that regional NOS activity is highly variable, and as was shown in some studies, no direct comparison can be made between the number of NADPHd-exhibiting, NOS immunoreactive and radioassay studies [21]. While many morphological studies analyzed the distribution of NADPHd-exhibiting and NOS immunoreactive neuronal pools, no such systemic studies are available with regard to the regional distribution of this functionally highly important enzyme [1,10,25–28]. Concerning the spinal cord, the occurrence of NADPHd-exhibiting and NOS immunoreactive neurons were found to occur in the superficial and deep dorsal horn, in the pericentral region all along the rostrocaudal axis of the spinal cord and in the IML extending from the upper thoracic to upper lumbar level. Comparative histochemical and immunocytochemical studies have confirmed this basic distribution of NOS immunoreactive neuronal pools in the spinal cord of different mammals including human [11,37,38]. Surprisingly, no such comprehensive studies are available concerning the NOS activity and therefore it is difficult to make an in-depth analysis with regard to changes in the NOS activity after various experimental interventions. The experiments performed in our laboratory clearly demonstrated regional and laminar differences in the activity of NOS in different experimental paradigms [22,32]. Similarly, the experiments performed recently studying ascending and descending propriospinal connections revealed along with a high number of nitrergic axons in the ventral and lateral columns an astonishingly high number of NADPHd-positive and NOS-IR stained neurons in the lumbosacral segments of the dog [24]. Surprisingly, segmental and laminar location of these somata strictly contradicts the findings presented recently by using the same segments and visualizing technique [41]. Comparing these results, a need emerged for mapping of cNOS activity all along the rostrocaudal axis of the spinal cord in the dog, beginning at the medulla–spinal cord junction to lower sacral segments, with the aim of detecting the regional and
ABSTRACT: The aim of this study was to examine the distribution of calcium-dependent nitric oxide synthase activity (cNOS) in the white and gray matter in cervical, thoracic, lumbar and sacral segments of the spinal cord and cauda equina of the dog. The enzyme’s activity, measured by the conversion of [3 H]arginine to [3 H]citrulline revealed considerable region-dependent differences along the rostrocaudal axis of the spinal cord in general and in cervical (C1, C2, C4, C6 and C8) and lumbar (L1–L3, L4–L7) segments in particular. In the non-compartmentalized spinal cord, the cNOS activity was lowest in the thoracic and highest in the sacral segments. No significant differences were noted in the gray matter regions (dorsal horn, intermediate zone and ventral horn) and the white matter columns (dorsal, lateral and ventral) in the upper cervical segments (C1–C4), except for a significant increase in the ventral horn of C4 segment. In C6 segment, the enzyme’s activity displayed significant differences in the intermediate zone, ventral and lateral columns. Surprisingly, extremely high cNOS activity was noted in the dorsal horn and dorsal column of the lowest cervical segment. Comparing the enzyme’s activity in upper and lower lumbar segments of the spinal cord, cNOS activity prevailed in L4–L7 segments in the dorsal horn and in all the above mentioned white matter columns. © 2002 Elsevier Science Inc. All rights reserved. KEY WORDS: cNOS Activity, Gray and white matter regions, Spinal cord, Dog.
INTRODUCTION Recently, neuronal nitric oxide synthase activity (NOS) has been shown to be constitutively present in some distinct loci in the CNS with high levels occurring in the cerebellum, phylogenetically old olfactory regions and the spinal cord. It has been known for a long time that a histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) can reveal one neuronal pool which is highly resistant to ischemia and excitotoxicity [35]. It has been simultaneously shown that NADPHd-exhibiting neurons are surviving in Parkinson and Huntington diseases and can be spared along with the somatostatin and neuropeptid Y neurons in different brain regions subjected to ischemia or anoxia [18–20,36]. In addition immunocytochemical staining for NOS has
∗ Address for correspondence: Dr. Nadežda Luk´ acˇ ov´a, Institute of Neurobiology, Slovak Academy of Sciences, Šolt´esovej 4, 040 01 Košice, Slovak Republic. Fax: +421-55-678-5074; E-mail: [email protected]
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174 laminar distribution of cNOS activity and comparing these values with fresh data for a quantitative and qualitative assessment of the spinal cord NOS neuronal pool. MATERIALS AND METHODS Surgical Procedure and Sample Dissection The experiments were performed on four adult dogs (n = 4) of either sex, weighing 5–8 kg. The experimental protocols were approved by the Institute of Neurobiology Animal Care Committee with the aim of minimizing both the suffering and the number of animals used. The animals were deeply anaesthetized with thiopental (50 mg/kg, i.v.) before the laminectomy was performed along the rostrocaudal axis of the spinal cord. The spinal cords were removed, put into ice-cold isotonic saline, cleaned from envelopes and carefully frozen in liquid nitrogen. The spinal cord segments (C1, C2, C4, C6, C8, Th1–Th12, L1–L3, L4–L7, S1–S3, and cauda equina) were cut by cryostat at −12◦ C into 600 µm slices. To identify the regional distribution of cNOS activity, the gray matter regions, i.e., dorsal horn (laminae I–VI), intermediate zone (laminae VII and X), ventral horn (laminae VIII–IX), and white matter divided into dorsal, lateral and ventral columns of C1, C2, C4, C6, C8, Th1–Th12, L1–L3, and L4–L7, were punched under a surgical microscope by needles (i.d. 0.6 and 0.8 mm) on a plate cooled with liquid nitrogen (−15◦ C). The white and gray matter of the S1–S3 segments were punched by the same technique and the cauda equina was taken as a whole. Radioassay Detection of cNOS Activity Calcium-dependent NOS activity was assayed by the conversion of [3 H]arginine to [3 H]citrulline according to the method of Bredt and Snyder [7] with a slight modification by Strosznajder and Chalimoniuk [34]. Frozen spinal cord samples were homogenized in 100–150 µl of an ice-cold Tris–HCl buffer (10 mM, pH = 7.4). The homogenates (200 µg/ml) were incubated for 45 min at 37◦ C with 10 µM [3 H]l-arginine (1 µCi), 1 mM NADPH, 1 µM calmodulin in 50 mM Hepes buffer, pH = 7.4 containing 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 100 µM flavin mononucleotide (FMN), 100 µM flavin adenine dinucleotide (FAD), 2 mM CaCl2 and 15 µM tetrahydrobiopterin (H4B) in a final volume of 300 µl. The reaction was stopped by the addition of 1 ml of ice-cold buffer which contained 10 mM EDTA and 100 mM Hepes buffer at pH = 5.5. Each sample was applied to a Dowex AG 50W-X8 cationic-exchange column (Na+ form) to remove [3 H]l-arginine. Columns were washed with 2 ml of deionized water to elute the [3 H]l-citrulline. Samples were centrifuged at 1000 × g for 5 min and aliquots (0.5 ml) of supernatant fractions were mixed with 5 ml of Bray’s fluid into scintillation vials and then counted in the Beckman LS-3801 scintillation counter. Cpms were converted to dpms using [3 H]-quenched standards. Levels of [3 H]l-citrulline were computed after subtracting the blank representing non-specific radioactivity in the absence of enzyme activity. Proteins were determined by the method of Bradford [5]. The enzyme activity was expressed as dpm/min/µg protein. Statistical Analysis The results of the radioassay detection of cNOS activity were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. RESULTS The results of the present study, obtained by the conversion of the [3 H]arginine to [3 H]citrulline point to a non-equal distribution
of cNOS activity along the rostrocaudal axis of the spinal cord of the dog and partly confirm our previous linked studies of the rabbit [21,31]. There were no significant differences in enzyme activity in the cervical and lumbar segments of the spinal cord and in the cauda equina. Contrary to this, significant differences were obtained in Th1–Th12 and S1–S3 segments, with the lowest level of cNOS activity observed in the thoracic (2.16±0.10 dpm/min/µg protein) and the highest (6.29±0.36 dpm/min/µg protein) in sacral segments of the spinal cord (Fig. 1). Only a low formation of radioactive products was detected in both gray and white matter of the Th1–Th12 segments. The enzyme activity clearly prevailed in the white matter of S1–S3 segments and in the cauda equina, when the results were compared to cervical ones (Table 1). Considerable differences in enzyme activity of the above mentioned spinal cord segments were detected in the dorsal horn, intermediate zone and ventral horn of the gray matter and in the white matter columns (Table 2). In comparison to the cervical segments, the cNOS activity was considerably lower in the dorsal horn, ventral horn, dorsal and lateral column of the thoracic spinal cord. The only significant differences found in cNOS activity in L1–L7 segments were 1.7- and 2.1-fold decreases in the ventral horn and dorsal column and a 98% increase in the lateral column. A comparison of the NOS activity through the segments of the cervical intumescence of the dog revealed no significant differences in the regions of C1, C2 and C4 segments, except for a significant increase in the ventral horn of C4 segment (Tables 3 and 4). However, noticeable differences were seen in the regions of the C6 segment. In the intermediate zone and in the ventral column of the white matter, the enzyme activity was significantly lower whereas that detected in the lateral column displayed a significantly higher cNOS activity. Surprisingly, a very high cNOS activity was found in the C8 segment, especially in the dorsal horn and dorsal column. The results presented in Table 5 compare the cNOS activity in the upper and lower lumbar segments of the spinal cord. A significantly higher level of cNOS activity was found in the dorsal horn and in all the white matter regions of the lower lumbar segments. It seems possible that exact segmental and regional analyses of cNOS activity undertaken within the control spinal cord, followed by experimental studies in close future, might enable an explanation of why a long-term inhibition of NOS activity causes the spinal cord infarcts just at the level of the cervicothoracic segments [4].
TABLE 1 THE DISTRIBUTION OF NITRIC OXIDE SYNTHASE ACTIVITY IN THE GRAY AND WHITE MATTER OF CERVICAL (C1–C8), THORACIC (TH1–TH12), LUMBAR (L1–L7), AND SACRAL (S1–S3) SEGMENTS OF THE SPINAL CORD AND IN CAUDA EQUINA (CE) OF THE DOG
Spinal cord segments C1–C8 Th1–Th12 L1–L7 S1–S3 CE
Gray matter (dpm/min/µg protein) 7.18 3.23 6.10 7.33 –
± ± ± ±
0.42 0.14* 0.55 0.45
White matter (dpm/min/µg protein) 2.21 1.09 1.73 5.25 4.22
± ± ± ± ±
0.16 0.06* 0.14 0.27* 0.27*
The enzyme activity was determined by the conversion of [3 H]arginine to [3 H]citrulline. The results were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. Data are the means of four experiments (n = 4), carried out in triplicate. ∗ p < 0.05 with respect to C1–C8 segments of each spinal cord region.
cNOS ACTIVITY IN THE SPINAL CORD OF THE DOG
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FIG. 1. The catalytic nitric oxide synthase (cNOS) activity in cervical (C1–C8), thoracic (Th1–Th12), lumbar (L1–L7), and sacral (S1–S3) segments of the spinal cord and in cauda equina (CE) of the dog. Catalytic NOS activity was detected by conversion of [3 H]arginine to [3 H]citrulline. The data are the means of four experiments. The results are expressed as dpm/min/µg protein, ∗ p < 0.05 with respect to C1–C8 segments.
TABLE 2 THE DISTRIBUTION OF NITRIC OXIDE SYNTHASE ACTIVITY IN THE GRAY MATTER REGIONS, AND IN THE WHITE MATTER COLUMNS1
Spinal cord regions Dorsal horn Intermediate zone Ventral horn Dorsal column Lateral column Ventral column
C1–C8 (dpm/min/µg protein) 8.80 6.54 6.20 2.85 2.22 1.56
± ± ± ± ± ±
Th1–Th12 (dpm/min/µg protein)
0.52 0.49 0.44 0.12 0.23 0.11
2.81 4.83 2.06 0.99 1.05 1.24
± ± ± ± ± ±
0.19* 0.18 0.07* 0.03* 0.04* 0.10
L1–L7 (dpm/min/µg protein) 8.59 5.99 3.72 1.39 4.40 1.60
± ± ± ± ± ±
0.48 0.74 0.43* 0.08* 0.24* 0.10
The enzyme activity was determined by the conversion of [3 H]arginine to [3 H]citrulline. The results were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. Data are the means of four experiments (n = 4), carried out in triplicate. 1 Gray matter regions refer to dorsal horn (laminae I–VI), intermediate zone (laminae VII and X), and ventral horn (laminae VIII–IX) while white matter columns refer to dorsal, lateral and ventral column in cervical (C1–C8), thoracic (Th1–Th12) and lumbar (L4–L7) spinal cord segments of the dog. ∗ p < 0.05 with respect to C1–C8 segments of each spinal cord region.
TABLE 3 NITRIC OXIDE SYNTHASE ACTIVITY IN GRAY MATTER REGIONS IN CERVICAL (C1, C2, C4, C6, AND C8) SEGMENTS OF THE SPINAL CORD
Segment C1 C2 C4 C6 C8
Dorsal horn (dpm/min/µg protein) 7.56 ± 0.44 6.89 ± 0.48 6.80 ± 0.11 6.01 ± 1.12 16.75 ± 0.43*
Intermediate zone (dpm/min/µg protein) 7.33 5.99 6.28 5.61 7.48
± ± ± ± ±
0.15 0.86 0.22 0.55* 0.71
Ventral horn (dpm/min/µg protein) 5.51 5.53 7.89 5.70 6.35
± ± ± ± ±
0.22 0.43 0.18* 0.24 0.23
Gray matter region refer to dorsal horn (laminae I–VI), intermediate zone (laminae VII and X), and ventral horn (laminae VIII–IX). The enzyme activity was determined by the conversion of [3 H]arginine to [3 H]citrulline. The results were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. Data are the means of four experiments (n = 4), carried out in triplicate. ∗ p < 0.05 with respect to C1 segment of each spinal cord region.
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176 TABLE 4
NITRIC OXIDE SYNTHASE ACTIVITY IN WHITE MATTER COLUMNS (DORSAL, LATERAL AND VENTRAL) IN CERVICAL (C1, C2, C4, C6, AND C8) SEGMENTS OF THE SPINAL CORD
Dorsal column (dpm/min/µg protein)
Segment C1 C2 C4 C6 C8
1.27 1.32 1.33 1.00 9.32
± ± ± ± ±
Lateral column (dpm/min/µg protein)
0.11 0.08 0.16 0.26 0.09*
2.50 2.04 2.76 1.77 2.05
± ± ± ± ±
0.04 0.23 0.36 0.16* 0.42
Ventral column (dpm/min/µg protein) 1.62 1.94 1.32 0.85 2.07
± ± ± ± ±
0.20 0.08 0.10 0.06* 0.10
The enzyme activity was determined by the conversion of [3 H]arginine to [3 H]citrulline. The results were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. Data are the means of four experiments (n = 4), carried out in triplicate. ∗ p < 0.05 with respect to C1 segment of each spinal cord region.
TABLE 5 NITRIC OXIDE SYNTHASE ACTIVITY IN THE GRAY MATTER REGIONS AND IN WHITE MATTER COLUMNS IN SEGMENTS OF UPPER (L1–L3) AND LOWER (L4–L7) LUMBAR INTUMESCENCE OF THE DOG
Spinal cord regions Dorsal horn Intermediate zone Ventral horn Dorsal column Lateral column Ventral column
L1–L3 (dpm/min/µg protein) 5.51 6.48 3.19 0.86 1.55 1.02
± ± ± ± ± ±
0.17 1.05 0.61 0.11 0.17 0.11
L4–L7 (dpm/min/µg protein) 11.68 5.50 4.24 1.92 2.84 2.19
± ± ± ± ± ±
0.80* 0.43 0.24 0.05* 0.31* 0.09*
Gray matter regions refer to dorsal horn (laminae I–VI), intermediate zone (laminae VII and X), and ventral horn (laminae VIII–IX) while white matter columns refer to dorsal, lateral and ventral column. The enzyme activity was determined by the conversion of [3 H]arginine to [3 H]citrulline. The results were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. Data are the means of four experiments (n = 4), carried out in triplicate. ∗ p < 0.05 with respect to L1–L3 segments of each spinal cord region.
DISCUSSION A comprehensive analysis of the distribution of the cNOS activity along the rostrocaudal axis of the spinal cord in the dog disclosed considerable differences among cervical, lumbar and sacral regions compared with the thoracic ones. It should be noted in this connection that the method used in our study allows to assess the calcium-dependent NOS activity in a given sample. However, this approach cannot explain possible differences in the regional distribution of endothelial and neuronal isoform of NOS activity in a given region. It also highlighted a remarkable incongruity between the cNOS activity in the gray and white matter in all regions studied. It should be noted that a radioassay detection of cNOS activity in given gray and white matter regions and segments (C1–S3) of the spinal cord reflects the occurrence of three different patterns of histochemically analyzed NADPHd-staining and/or NOS-immunoreactivity in (a) cell bodies, (b) the fiber-like expression of the occurrence of nNOS in axons and dendrites, and (c) a highly important punctate, non-somatic NADPHd-staining or NOS immunoreactivity seen with varying intensity in the neuropil of various gray matter regions all along the rostrocaudal axis of the spinal cord. From a quantitative point of view, the second and third patterns are of special importance because biochemical studies have indicated that nNOS partitions largely with membrane-associated subcellular fractions [8,15] and electron microscopy has identified
>80% of nNOS immunoreactivity in monkey visual cortex as axonal and dendritic profiles [2]. Even more importantly, the ratio of three components of NOS immunostaining varies from region to region and from segment to segment. This variation makes a precise biochemical assessment of cNOS activity extremely difficult even with the use of normal control material. In order to find an explanation for the variations, we shall first consider a significantly lower cNOS activity detected in the gray matter of the thoracic region in comparison with the cervical and lumbosacral ones and then we will look at the cNOS activity in the white matter of the corresponding regions. Remarkable differences were found analyzing the compartmentalized gray matter of the cervical, thoracic and lumbar segments in the dorsal horn, intermediate zone and ventral horn in general and among individual cervical, upper and lower lumbar segments in particular. Considering a massive occurrence of the fiber-like, and punctate non-somatic nNOS staining in the superficial and deep dorsal horn and pericentral region in the cervical, lumbar and sacral regions detected in recent studies, it seems conceivable that the bulk of neuronal NOS activity assessed in these regions may result mainly from the punctate NADPHd histochemical and NOS immunostaining [17,22–28]. This view is supported by the low density of the punctate NADPHd histochemical staining and NOS immunostaining seen across the superficial dorsal horn of all thoracic segments with the exception of Th1. It is possible that lower levels of cNOS activity in the dorsal horn of Th1–Th12 segments in comparison with the same compartment seen in C1–C8 and L1–L7 segments are merely possible due to a small extent of the dorsal horn per se accompanied by a quantitatively low number of NOS immunoreactive neurons throughout the thoracic segments and a greatly reduced fiber-like and punctate NOS immunoreactivity seen therein. It should be mentioned that the compartmentalized gray matter of the thoracic segments displayed a consistently higher level of cNOS activity in the intermediate zone, a finding caused by an accumulation of large, highly NOS immunoreactive and NADPHd-exhibiting neurons in the intermediolateral cell column (IML) and intercalate nucleus, the latter forming a more medially located structure belonging, along with IML, to the thoracic sympathetic system. An unexpectedly high level of NOS activity was found in the compartmentalized ventral horn in all cervical segments when taken together. It is interesting to note that almost all recent histochemical and immunohistochemical studies have described the occurrence of NOS immunoreactive neurons in ventral horn as strictly limited in number or as virtually absent. However, detailed studies performed in our laboratory using carefully prepared perfusion fixed material of the dog have clearly demonstrated the presence of NOS immunoreactive neurons in the ventral horn mainly in its dorsomedial area (lamina VIII) and in the adjoining
cNOS ACTIVITY IN THE SPINAL CORD OF THE DOG part of the intermediate zone (lamina VII) [30,31]. With regard to lamina VIII, a high number of NOS-immunoreactive neurons could be found mainly in the cervical and lumbosacral enlargements. This finding is supported by a recent histochemical study of NADPHd-exhibiting neurons occurring in the early phase of ontogenesis of the human spinal cord, topographically located in the medial and dorsomedial part of the ventral horn and considered to be a part of an interneuronal pool [12,13]. An intriguing finding was found when the cervical and lumbar segments were considered separately. The results revealed enormously high levels of cNOS activity in the dorsal horn of C8 segment forming a part of the cervical enlargement and of L4–L7 segments occurring in the extent of the lumbosacral enlargement. As the number of NOS immunoreactive neurons in the dorsal horn of both spinal cord regions is not dramatically different from that found in the neighboring segments, the cell bodies located there can scarcely lead to an enormous increase of cNOS activity in both areas. An extrinsic source of NOS-containing fibers entering the dorsal horn via the dorsal root afferents and having parent NOS-immunoreactive cell bodies in the corresponding dorsal root ganglia is the likely cause of the increased cNOS activity in C8 and L4–L7 dorsal horn. Even though a lower level of cNOS activity was found in the white matter in comparison with the gray one all along the rostrocaudal axis, the occurrence of cNOS activity in the white matter appeared to be an interesting finding worthy of a more detailed examination. It has been well established that NOS-containing somata are lacking in the white matter of the spinal cord and, therefore, they cannot add to the cNOS activity observed in this compartment. Moreover, white matter is a non-synaptic compartment that completely lacks punctate NADPHd staining or NOS punctate immunoreactivity and, as a consequence, cNOS activity detected in the extent of the spinal cord white matter may result solely from axonal NOS which thus appears to be an orthogradely transportable enzyme synthesized in neurons having short- or long-projection axons passing for various distances from parent cell body alone or colocalized with another neurotransmitter and reaching a more or less distant neuronal pools. This view is consistent with our previous finding of the presence of many NADPHd-exhibiting and NOS immunoreactive axons in the three white matter columns in different segments of the rabbit and dog spinal cord [26–28]. The occurrence of NOS immunoreactive axons in the white matter have been mentioned sporadically [29,37,41] but no systematic study has been made as yet to describe the nitrergic connections although such descriptions exist for dopaminergic, serotoninergic, noradrenergic and cholinergic pathways and their distinct location and functional implications in the spinal cord circuitry [3,9,14,16,33]. Higher cNOS activity in the white matter of the sacral spinal cord in comparison to the cervical one seems to be in keeping with a large number of NOS immunoreactive axons, including very thick ones, not only in the ventral and lateral columns but also seen, especially, as very thin NOS immunoreactive axonal profiles in the dorsal column close to the dorsal sacral commissure. ACKNOWLEDGEMENTS
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PII: S0361-9230(02)00774-8
The regional distribution of nitric oxide synthase activity in the spinal cord of the dog 1∗ Dáša C´ ˇ ıžková,1 Martin Maršala,2 Imrich Lukácˇ 3 and Jozef Maršala1 ˇ Nadežda Lukácová, 1 Institute
of Neurobiology, Slovak Academy of Sciences, Košice, Slovak Republic; 2 Anesthesiology Research Laboratory, 0818 University of California, La Jolla, CA, USA; and 3 Department of Neurosurgery, Medical Faculty of P. J. Šafárik University, Košice, Slovak Republic [Received 7 January 2002; Accepted 12 February 2002] been developed and it was clearly demonstrated that this staining is almost identical to histochemical staining for NADPHd. Therefore, both these different techniques are currently used to localize the neurons that are small in number and considered to be not more that 1–2% of total neuronal population [6]. However, the number of NADPHd-exhibiting and NOS immunoreactive neurons was found to be regionally different [39,40]. Moreover, biochemical analysis of cNOS activity documented that regional NOS activity is highly variable, and as was shown in some studies, no direct comparison can be made between the number of NADPHd-exhibiting, NOS immunoreactive and radioassay studies [21]. While many morphological studies analyzed the distribution of NADPHd-exhibiting and NOS immunoreactive neuronal pools, no such systemic studies are available with regard to the regional distribution of this functionally highly important enzyme [1,10,25–28]. Concerning the spinal cord, the occurrence of NADPHd-exhibiting and NOS immunoreactive neurons were found to occur in the superficial and deep dorsal horn, in the pericentral region all along the rostrocaudal axis of the spinal cord and in the IML extending from the upper thoracic to upper lumbar level. Comparative histochemical and immunocytochemical studies have confirmed this basic distribution of NOS immunoreactive neuronal pools in the spinal cord of different mammals including human [11,37,38]. Surprisingly, no such comprehensive studies are available concerning the NOS activity and therefore it is difficult to make an in-depth analysis with regard to changes in the NOS activity after various experimental interventions. The experiments performed in our laboratory clearly demonstrated regional and laminar differences in the activity of NOS in different experimental paradigms [22,32]. Similarly, the experiments performed recently studying ascending and descending propriospinal connections revealed along with a high number of nitrergic axons in the ventral and lateral columns an astonishingly high number of NADPHd-positive and NOS-IR stained neurons in the lumbosacral segments of the dog [24]. Surprisingly, segmental and laminar location of these somata strictly contradicts the findings presented recently by using the same segments and visualizing technique [41]. Comparing these results, a need emerged for mapping of cNOS activity all along the rostrocaudal axis of the spinal cord in the dog, beginning at the medulla–spinal cord junction to lower sacral segments, with the aim of detecting the regional and
ABSTRACT: The aim of this study was to examine the distribution of calcium-dependent nitric oxide synthase activity (cNOS) in the white and gray matter in cervical, thoracic, lumbar and sacral segments of the spinal cord and cauda equina of the dog. The enzyme’s activity, measured by the conversion of [3 H]arginine to [3 H]citrulline revealed considerable region-dependent differences along the rostrocaudal axis of the spinal cord in general and in cervical (C1, C2, C4, C6 and C8) and lumbar (L1–L3, L4–L7) segments in particular. In the non-compartmentalized spinal cord, the cNOS activity was lowest in the thoracic and highest in the sacral segments. No significant differences were noted in the gray matter regions (dorsal horn, intermediate zone and ventral horn) and the white matter columns (dorsal, lateral and ventral) in the upper cervical segments (C1–C4), except for a significant increase in the ventral horn of C4 segment. In C6 segment, the enzyme’s activity displayed significant differences in the intermediate zone, ventral and lateral columns. Surprisingly, extremely high cNOS activity was noted in the dorsal horn and dorsal column of the lowest cervical segment. Comparing the enzyme’s activity in upper and lower lumbar segments of the spinal cord, cNOS activity prevailed in L4–L7 segments in the dorsal horn and in all the above mentioned white matter columns. © 2002 Elsevier Science Inc. All rights reserved. KEY WORDS: cNOS Activity, Gray and white matter regions, Spinal cord, Dog.
INTRODUCTION Recently, neuronal nitric oxide synthase activity (NOS) has been shown to be constitutively present in some distinct loci in the CNS with high levels occurring in the cerebellum, phylogenetically old olfactory regions and the spinal cord. It has been known for a long time that a histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) can reveal one neuronal pool which is highly resistant to ischemia and excitotoxicity [35]. It has been simultaneously shown that NADPHd-exhibiting neurons are surviving in Parkinson and Huntington diseases and can be spared along with the somatostatin and neuropeptid Y neurons in different brain regions subjected to ischemia or anoxia [18–20,36]. In addition immunocytochemical staining for NOS has
∗ Address for correspondence: Dr. Nadežda Luk´ acˇ ov´a, Institute of Neurobiology, Slovak Academy of Sciences, Šolt´esovej 4, 040 01 Košice, Slovak Republic. Fax: +421-55-678-5074; E-mail: [email protected]
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174 laminar distribution of cNOS activity and comparing these values with fresh data for a quantitative and qualitative assessment of the spinal cord NOS neuronal pool. MATERIALS AND METHODS Surgical Procedure and Sample Dissection The experiments were performed on four adult dogs (n = 4) of either sex, weighing 5–8 kg. The experimental protocols were approved by the Institute of Neurobiology Animal Care Committee with the aim of minimizing both the suffering and the number of animals used. The animals were deeply anaesthetized with thiopental (50 mg/kg, i.v.) before the laminectomy was performed along the rostrocaudal axis of the spinal cord. The spinal cords were removed, put into ice-cold isotonic saline, cleaned from envelopes and carefully frozen in liquid nitrogen. The spinal cord segments (C1, C2, C4, C6, C8, Th1–Th12, L1–L3, L4–L7, S1–S3, and cauda equina) were cut by cryostat at −12◦ C into 600 µm slices. To identify the regional distribution of cNOS activity, the gray matter regions, i.e., dorsal horn (laminae I–VI), intermediate zone (laminae VII and X), ventral horn (laminae VIII–IX), and white matter divided into dorsal, lateral and ventral columns of C1, C2, C4, C6, C8, Th1–Th12, L1–L3, and L4–L7, were punched under a surgical microscope by needles (i.d. 0.6 and 0.8 mm) on a plate cooled with liquid nitrogen (−15◦ C). The white and gray matter of the S1–S3 segments were punched by the same technique and the cauda equina was taken as a whole. Radioassay Detection of cNOS Activity Calcium-dependent NOS activity was assayed by the conversion of [3 H]arginine to [3 H]citrulline according to the method of Bredt and Snyder [7] with a slight modification by Strosznajder and Chalimoniuk [34]. Frozen spinal cord samples were homogenized in 100–150 µl of an ice-cold Tris–HCl buffer (10 mM, pH = 7.4). The homogenates (200 µg/ml) were incubated for 45 min at 37◦ C with 10 µM [3 H]l-arginine (1 µCi), 1 mM NADPH, 1 µM calmodulin in 50 mM Hepes buffer, pH = 7.4 containing 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 100 µM flavin mononucleotide (FMN), 100 µM flavin adenine dinucleotide (FAD), 2 mM CaCl2 and 15 µM tetrahydrobiopterin (H4B) in a final volume of 300 µl. The reaction was stopped by the addition of 1 ml of ice-cold buffer which contained 10 mM EDTA and 100 mM Hepes buffer at pH = 5.5. Each sample was applied to a Dowex AG 50W-X8 cationic-exchange column (Na+ form) to remove [3 H]l-arginine. Columns were washed with 2 ml of deionized water to elute the [3 H]l-citrulline. Samples were centrifuged at 1000 × g for 5 min and aliquots (0.5 ml) of supernatant fractions were mixed with 5 ml of Bray’s fluid into scintillation vials and then counted in the Beckman LS-3801 scintillation counter. Cpms were converted to dpms using [3 H]-quenched standards. Levels of [3 H]l-citrulline were computed after subtracting the blank representing non-specific radioactivity in the absence of enzyme activity. Proteins were determined by the method of Bradford [5]. The enzyme activity was expressed as dpm/min/µg protein. Statistical Analysis The results of the radioassay detection of cNOS activity were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. RESULTS The results of the present study, obtained by the conversion of the [3 H]arginine to [3 H]citrulline point to a non-equal distribution
of cNOS activity along the rostrocaudal axis of the spinal cord of the dog and partly confirm our previous linked studies of the rabbit [21,31]. There were no significant differences in enzyme activity in the cervical and lumbar segments of the spinal cord and in the cauda equina. Contrary to this, significant differences were obtained in Th1–Th12 and S1–S3 segments, with the lowest level of cNOS activity observed in the thoracic (2.16±0.10 dpm/min/µg protein) and the highest (6.29±0.36 dpm/min/µg protein) in sacral segments of the spinal cord (Fig. 1). Only a low formation of radioactive products was detected in both gray and white matter of the Th1–Th12 segments. The enzyme activity clearly prevailed in the white matter of S1–S3 segments and in the cauda equina, when the results were compared to cervical ones (Table 1). Considerable differences in enzyme activity of the above mentioned spinal cord segments were detected in the dorsal horn, intermediate zone and ventral horn of the gray matter and in the white matter columns (Table 2). In comparison to the cervical segments, the cNOS activity was considerably lower in the dorsal horn, ventral horn, dorsal and lateral column of the thoracic spinal cord. The only significant differences found in cNOS activity in L1–L7 segments were 1.7- and 2.1-fold decreases in the ventral horn and dorsal column and a 98% increase in the lateral column. A comparison of the NOS activity through the segments of the cervical intumescence of the dog revealed no significant differences in the regions of C1, C2 and C4 segments, except for a significant increase in the ventral horn of C4 segment (Tables 3 and 4). However, noticeable differences were seen in the regions of the C6 segment. In the intermediate zone and in the ventral column of the white matter, the enzyme activity was significantly lower whereas that detected in the lateral column displayed a significantly higher cNOS activity. Surprisingly, a very high cNOS activity was found in the C8 segment, especially in the dorsal horn and dorsal column. The results presented in Table 5 compare the cNOS activity in the upper and lower lumbar segments of the spinal cord. A significantly higher level of cNOS activity was found in the dorsal horn and in all the white matter regions of the lower lumbar segments. It seems possible that exact segmental and regional analyses of cNOS activity undertaken within the control spinal cord, followed by experimental studies in close future, might enable an explanation of why a long-term inhibition of NOS activity causes the spinal cord infarcts just at the level of the cervicothoracic segments [4].
TABLE 1 THE DISTRIBUTION OF NITRIC OXIDE SYNTHASE ACTIVITY IN THE GRAY AND WHITE MATTER OF CERVICAL (C1–C8), THORACIC (TH1–TH12), LUMBAR (L1–L7), AND SACRAL (S1–S3) SEGMENTS OF THE SPINAL CORD AND IN CAUDA EQUINA (CE) OF THE DOG
Spinal cord segments C1–C8 Th1–Th12 L1–L7 S1–S3 CE
Gray matter (dpm/min/µg protein) 7.18 3.23 6.10 7.33 –
± ± ± ±
0.42 0.14* 0.55 0.45
White matter (dpm/min/µg protein) 2.21 1.09 1.73 5.25 4.22
± ± ± ± ±
0.16 0.06* 0.14 0.27* 0.27*
The enzyme activity was determined by the conversion of [3 H]arginine to [3 H]citrulline. The results were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. Data are the means of four experiments (n = 4), carried out in triplicate. ∗ p < 0.05 with respect to C1–C8 segments of each spinal cord region.
cNOS ACTIVITY IN THE SPINAL CORD OF THE DOG
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FIG. 1. The catalytic nitric oxide synthase (cNOS) activity in cervical (C1–C8), thoracic (Th1–Th12), lumbar (L1–L7), and sacral (S1–S3) segments of the spinal cord and in cauda equina (CE) of the dog. Catalytic NOS activity was detected by conversion of [3 H]arginine to [3 H]citrulline. The data are the means of four experiments. The results are expressed as dpm/min/µg protein, ∗ p < 0.05 with respect to C1–C8 segments.
TABLE 2 THE DISTRIBUTION OF NITRIC OXIDE SYNTHASE ACTIVITY IN THE GRAY MATTER REGIONS, AND IN THE WHITE MATTER COLUMNS1
Spinal cord regions Dorsal horn Intermediate zone Ventral horn Dorsal column Lateral column Ventral column
C1–C8 (dpm/min/µg protein) 8.80 6.54 6.20 2.85 2.22 1.56
± ± ± ± ± ±
Th1–Th12 (dpm/min/µg protein)
0.52 0.49 0.44 0.12 0.23 0.11
2.81 4.83 2.06 0.99 1.05 1.24
± ± ± ± ± ±
0.19* 0.18 0.07* 0.03* 0.04* 0.10
L1–L7 (dpm/min/µg protein) 8.59 5.99 3.72 1.39 4.40 1.60
± ± ± ± ± ±
0.48 0.74 0.43* 0.08* 0.24* 0.10
The enzyme activity was determined by the conversion of [3 H]arginine to [3 H]citrulline. The results were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. Data are the means of four experiments (n = 4), carried out in triplicate. 1 Gray matter regions refer to dorsal horn (laminae I–VI), intermediate zone (laminae VII and X), and ventral horn (laminae VIII–IX) while white matter columns refer to dorsal, lateral and ventral column in cervical (C1–C8), thoracic (Th1–Th12) and lumbar (L4–L7) spinal cord segments of the dog. ∗ p < 0.05 with respect to C1–C8 segments of each spinal cord region.
TABLE 3 NITRIC OXIDE SYNTHASE ACTIVITY IN GRAY MATTER REGIONS IN CERVICAL (C1, C2, C4, C6, AND C8) SEGMENTS OF THE SPINAL CORD
Segment C1 C2 C4 C6 C8
Dorsal horn (dpm/min/µg protein) 7.56 ± 0.44 6.89 ± 0.48 6.80 ± 0.11 6.01 ± 1.12 16.75 ± 0.43*
Intermediate zone (dpm/min/µg protein) 7.33 5.99 6.28 5.61 7.48
± ± ± ± ±
0.15 0.86 0.22 0.55* 0.71
Ventral horn (dpm/min/µg protein) 5.51 5.53 7.89 5.70 6.35
± ± ± ± ±
0.22 0.43 0.18* 0.24 0.23
Gray matter region refer to dorsal horn (laminae I–VI), intermediate zone (laminae VII and X), and ventral horn (laminae VIII–IX). The enzyme activity was determined by the conversion of [3 H]arginine to [3 H]citrulline. The results were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. Data are the means of four experiments (n = 4), carried out in triplicate. ∗ p < 0.05 with respect to C1 segment of each spinal cord region.
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176 TABLE 4
NITRIC OXIDE SYNTHASE ACTIVITY IN WHITE MATTER COLUMNS (DORSAL, LATERAL AND VENTRAL) IN CERVICAL (C1, C2, C4, C6, AND C8) SEGMENTS OF THE SPINAL CORD
Dorsal column (dpm/min/µg protein)
Segment C1 C2 C4 C6 C8
1.27 1.32 1.33 1.00 9.32
± ± ± ± ±
Lateral column (dpm/min/µg protein)
0.11 0.08 0.16 0.26 0.09*
2.50 2.04 2.76 1.77 2.05
± ± ± ± ±
0.04 0.23 0.36 0.16* 0.42
Ventral column (dpm/min/µg protein) 1.62 1.94 1.32 0.85 2.07
± ± ± ± ±
0.20 0.08 0.10 0.06* 0.10
The enzyme activity was determined by the conversion of [3 H]arginine to [3 H]citrulline. The results were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. Data are the means of four experiments (n = 4), carried out in triplicate. ∗ p < 0.05 with respect to C1 segment of each spinal cord region.
TABLE 5 NITRIC OXIDE SYNTHASE ACTIVITY IN THE GRAY MATTER REGIONS AND IN WHITE MATTER COLUMNS IN SEGMENTS OF UPPER (L1–L3) AND LOWER (L4–L7) LUMBAR INTUMESCENCE OF THE DOG
Spinal cord regions Dorsal horn Intermediate zone Ventral horn Dorsal column Lateral column Ventral column
L1–L3 (dpm/min/µg protein) 5.51 6.48 3.19 0.86 1.55 1.02
± ± ± ± ± ±
0.17 1.05 0.61 0.11 0.17 0.11
L4–L7 (dpm/min/µg protein) 11.68 5.50 4.24 1.92 2.84 2.19
± ± ± ± ± ±
0.80* 0.43 0.24 0.05* 0.31* 0.09*
Gray matter regions refer to dorsal horn (laminae I–VI), intermediate zone (laminae VII and X), and ventral horn (laminae VIII–IX) while white matter columns refer to dorsal, lateral and ventral column. The enzyme activity was determined by the conversion of [3 H]arginine to [3 H]citrulline. The results were statistically evaluated by ANOVA as well as by the Tukey–Kramer test and have been given as means ± SEM. Data are the means of four experiments (n = 4), carried out in triplicate. ∗ p < 0.05 with respect to L1–L3 segments of each spinal cord region.
DISCUSSION A comprehensive analysis of the distribution of the cNOS activity along the rostrocaudal axis of the spinal cord in the dog disclosed considerable differences among cervical, lumbar and sacral regions compared with the thoracic ones. It should be noted in this connection that the method used in our study allows to assess the calcium-dependent NOS activity in a given sample. However, this approach cannot explain possible differences in the regional distribution of endothelial and neuronal isoform of NOS activity in a given region. It also highlighted a remarkable incongruity between the cNOS activity in the gray and white matter in all regions studied. It should be noted that a radioassay detection of cNOS activity in given gray and white matter regions and segments (C1–S3) of the spinal cord reflects the occurrence of three different patterns of histochemically analyzed NADPHd-staining and/or NOS-immunoreactivity in (a) cell bodies, (b) the fiber-like expression of the occurrence of nNOS in axons and dendrites, and (c) a highly important punctate, non-somatic NADPHd-staining or NOS immunoreactivity seen with varying intensity in the neuropil of various gray matter regions all along the rostrocaudal axis of the spinal cord. From a quantitative point of view, the second and third patterns are of special importance because biochemical studies have indicated that nNOS partitions largely with membrane-associated subcellular fractions [8,15] and electron microscopy has identified
>80% of nNOS immunoreactivity in monkey visual cortex as axonal and dendritic profiles [2]. Even more importantly, the ratio of three components of NOS immunostaining varies from region to region and from segment to segment. This variation makes a precise biochemical assessment of cNOS activity extremely difficult even with the use of normal control material. In order to find an explanation for the variations, we shall first consider a significantly lower cNOS activity detected in the gray matter of the thoracic region in comparison with the cervical and lumbosacral ones and then we will look at the cNOS activity in the white matter of the corresponding regions. Remarkable differences were found analyzing the compartmentalized gray matter of the cervical, thoracic and lumbar segments in the dorsal horn, intermediate zone and ventral horn in general and among individual cervical, upper and lower lumbar segments in particular. Considering a massive occurrence of the fiber-like, and punctate non-somatic nNOS staining in the superficial and deep dorsal horn and pericentral region in the cervical, lumbar and sacral regions detected in recent studies, it seems conceivable that the bulk of neuronal NOS activity assessed in these regions may result mainly from the punctate NADPHd histochemical and NOS immunostaining [17,22–28]. This view is supported by the low density of the punctate NADPHd histochemical staining and NOS immunostaining seen across the superficial dorsal horn of all thoracic segments with the exception of Th1. It is possible that lower levels of cNOS activity in the dorsal horn of Th1–Th12 segments in comparison with the same compartment seen in C1–C8 and L1–L7 segments are merely possible due to a small extent of the dorsal horn per se accompanied by a quantitatively low number of NOS immunoreactive neurons throughout the thoracic segments and a greatly reduced fiber-like and punctate NOS immunoreactivity seen therein. It should be mentioned that the compartmentalized gray matter of the thoracic segments displayed a consistently higher level of cNOS activity in the intermediate zone, a finding caused by an accumulation of large, highly NOS immunoreactive and NADPHd-exhibiting neurons in the intermediolateral cell column (IML) and intercalate nucleus, the latter forming a more medially located structure belonging, along with IML, to the thoracic sympathetic system. An unexpectedly high level of NOS activity was found in the compartmentalized ventral horn in all cervical segments when taken together. It is interesting to note that almost all recent histochemical and immunohistochemical studies have described the occurrence of NOS immunoreactive neurons in ventral horn as strictly limited in number or as virtually absent. However, detailed studies performed in our laboratory using carefully prepared perfusion fixed material of the dog have clearly demonstrated the presence of NOS immunoreactive neurons in the ventral horn mainly in its dorsomedial area (lamina VIII) and in the adjoining
cNOS ACTIVITY IN THE SPINAL CORD OF THE DOG part of the intermediate zone (lamina VII) [30,31]. With regard to lamina VIII, a high number of NOS-immunoreactive neurons could be found mainly in the cervical and lumbosacral enlargements. This finding is supported by a recent histochemical study of NADPHd-exhibiting neurons occurring in the early phase of ontogenesis of the human spinal cord, topographically located in the medial and dorsomedial part of the ventral horn and considered to be a part of an interneuronal pool [12,13]. An intriguing finding was found when the cervical and lumbar segments were considered separately. The results revealed enormously high levels of cNOS activity in the dorsal horn of C8 segment forming a part of the cervical enlargement and of L4–L7 segments occurring in the extent of the lumbosacral enlargement. As the number of NOS immunoreactive neurons in the dorsal horn of both spinal cord regions is not dramatically different from that found in the neighboring segments, the cell bodies located there can scarcely lead to an enormous increase of cNOS activity in both areas. An extrinsic source of NOS-containing fibers entering the dorsal horn via the dorsal root afferents and having parent NOS-immunoreactive cell bodies in the corresponding dorsal root ganglia is the likely cause of the increased cNOS activity in C8 and L4–L7 dorsal horn. Even though a lower level of cNOS activity was found in the white matter in comparison with the gray one all along the rostrocaudal axis, the occurrence of cNOS activity in the white matter appeared to be an interesting finding worthy of a more detailed examination. It has been well established that NOS-containing somata are lacking in the white matter of the spinal cord and, therefore, they cannot add to the cNOS activity observed in this compartment. Moreover, white matter is a non-synaptic compartment that completely lacks punctate NADPHd staining or NOS punctate immunoreactivity and, as a consequence, cNOS activity detected in the extent of the spinal cord white matter may result solely from axonal NOS which thus appears to be an orthogradely transportable enzyme synthesized in neurons having short- or long-projection axons passing for various distances from parent cell body alone or colocalized with another neurotransmitter and reaching a more or less distant neuronal pools. This view is consistent with our previous finding of the presence of many NADPHd-exhibiting and NOS immunoreactive axons in the three white matter columns in different segments of the rabbit and dog spinal cord [26–28]. The occurrence of NOS immunoreactive axons in the white matter have been mentioned sporadically [29,37,41] but no systematic study has been made as yet to describe the nitrergic connections although such descriptions exist for dopaminergic, serotoninergic, noradrenergic and cholinergic pathways and their distinct location and functional implications in the spinal cord circuitry [3,9,14,16,33]. Higher cNOS activity in the white matter of the sacral spinal cord in comparison to the cervical one seems to be in keeping with a large number of NOS immunoreactive axons, including very thick ones, not only in the ventral and lateral columns but also seen, especially, as very thin NOS immunoreactive axonal profiles in the dorsal column close to the dorsal sacral commissure. ACKNOWLEDGEMENTS
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