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Sympathetic postganglionic pathways to the hind-limb of the rat: a fluorescent histochemical study
Journal of the Autonomic Nervous System. 13 (1985) 171-174
171
Elsevier JAN 00440
Short Communications
Sympathetic postganglionic pathways to the hind-limb of the rat: a fluorescent histochemical study D. Fred Peterson and John E. Norvell Departments of Physiology and Anatomy, Oral Roberts UniversiO' Schools of Medicine and Dentistry, Tulsa, OK 74171 (U.S.A.)
(Received November 13th, 1984) (Revised version receivedJanuary 25th, 1985) (Accepted February 4th, 1985)
K e y words: sympathectomy - autonomic nerves - lumbar sympathetic chain
Our findings indicate that, in the rat, (1) 90% of sympathetic postganglionic fibers innervating the femoral arteries originate in or pass through the L 3 - L 4 sympathetic chain ganglia and (2) a small amount of contralateral innervation is likely in the lumbar region. Sympathetic innervation of resistance vessels in mammals contributes to distribution of blood flow in exercise [1]. In order to study sympathetic influences on blood flow in conscious animals, alteration of sympathetic activity must be accomplished without interfering with motor control of the exercising muscle. This has been accomplished in the dog by extensive lumbar sympathectomy [6]. Because of the small size of the rat and the possibility that surgical trauma would interfere with normal exercise, more limited sympathectomy, if possible, may be necessary in this species. The present study was designed to determine the course of sympathetic postganglionic fibers in the lumbar sympathetic chain supplying the femoral arteries. Its purpose was to identify the minimal denervation necessary to interrupt sympathetic outflow to the hind-limbs of the rat. Forty-three male Sprague-Dawley rats (350-500 g) underwent surgery to remove a section of the abdominal sympathetic trunk. Sham operations were performed on 5 additional rats under sodium pentobarbital anesthesia (30 m g / k g ) , a left abdominal incision was made above the kidney. The aorta was reflected ventrally and left psoas muscles reflected laterally thus exposing the sympathetic chains.
Correspondence: D.F. Peterson, Department of Physiology,Oral Roberts University School of Medicine, Tulsa, OK 74171, U.S.A.
0165-1838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
172 Sections of one or both chains and associated ganglia between T I 3 and L5 were then removed and the incision was sutured closed. Each rat was given a single dose of 30,000 units of penicillin (Bicillin, Wyeth) and allowed to recover between 6 - 1 7 days. The histofluorescence technique of De la Torre [5], utilizing glyoxylic acid was employed to determine the presence of catecholamines in the adventitia of the femoral artery and thus estimate the extent of interruption of postganglionic pathways to the hind-limb. Rats were decapitated and the pectineus muscle and associated femoral artery removed from both hind-legs. Heart muscle (atria) was also removed as a reference. The tissue was quick-frozen in 2-methylbutane cooled by liquid nitrogen and 20/~m sections were cut and treated. Sections were examined and photographed under fluorescence microscopy. If the atrial tissue did not indicate extensive presence of catecholamine, the animal was rejected. Sections containing the femoral artery were subjectively graded for fluorescence intensity on a scale of 0 - 3 . The grader did not k n o w the extent of the s y m p a t h e c t o m y at the time of grading. Zero indicated the absence of fluorescence and 3 indicated m a x i m u m intensity. Table I compares the intensity of fluorescence in the right and left femoral arteries to the extent of lumbar sympathectomy. It is apparent that when the left chain was removed from, and including the L 1 - L 5 ganglia that there was major loss of fluorescence in the left leg. In contrast, fluorescence in the right leg, did not indicate a significant reduction (Table I). W h e n L 3 - L 4 were removed on the left side, left femoral fluorescence was significantly reduced ( - 59%), but it was not as impressive as after L 1 - L 5 removals. Right side fluorescence tended to be reduced but it did not prove to be significant. Removal of T 1 3 - L 2 significantly reduced fluorescence on the left side but did not affect the right side.
TABLE I PRESENCE OF CATECHOLAMINE FLUORESCENCE IN THE FEMORAL ARTERY AFTER SELECTIVE REMOVAL OF SYMPATHETIC CHAINS Values represent averaged intensities (mean + S.E.M.) of fluorescence with 3 = maximum. B/S, sections of both chains removed; L, sections of left chain removed. Statistics performed were a one way analysis of variance and Duncan's multi-range test, Unbroken vertical bars indicate no significant difference exists between inclusive values. For example, under Left femoral, B/S L3-L4 is significantly different from both control and B/S L4-L5 but not significantly different from any other values. Control B/S L4-L5 L L3-L4 L T13-L2 B/S L2 B/S L2-L3 L L1-L5 B/S L3-L4
Left femoral
(n)
2.30 + 0.34 1.50+0.50 0.94 + 0.29 0.90+0.47 0.70 + 0.49 0.50-t-0.33 0.37+0.43 0.24+0.15
5 5 8
4 5 4 4 11
Control L T13-L2 L El-L5 L L3-L4 B/S IA-L5 B/S L2 B/S L2-L3 B/S L3-L4
Right femoral
(n)
2.80 + 0.14 [ 2.63 + 0.45 ] 2.38 + 0.301 2.33+0.291 1.40+0.45 ] 1.40 ± 0.45 0.80+0.52 I 0.27+0.08 [
5 4 4 9 5 5 5 11
173
The dramatic difference between ipsilateral and contralateral femoral fluorescence after unilateral denervation suggests strong dominance of ipsilateral sympathetic innervation to the hindqimb in rats. This has been assumed to be true in earlier studies on dogs [6,8]. On the other hand, since some fluorescence persisted on the ipsilateral side, our data suggest that there may be a small amount of cross-over from the contralateral lumbar sympathetic chain. The effect of bilateral removal of pairs of sympathetic chain ganglia was compared with unilateral removal (Table I). Bilateral removal of the L4-L5 ganglia caused 35-50% reduction in fluorescence. Bilateral removal of the L 3 - L 4 reduced fluorescence by 90% in both hind-legs, whereas removal of L 2 - L 3 reduced fluorescence by 71-78%. Finally, if the L2 ganglia were both removed, 50-70% reduction in fluorescence was observed. From a left side incision intended as a chronic surgical procedure in exercise studies, we found it technically difficult to locate and remove the right L1 ganglion without damaging the crural ligament. In fact, the right L2 ganglion is ordinarily not visible and is found by tracing the right sympathetic chain as it passes through folds of the ligament. Hence, we did not attempt bilateral denervation above the L2 level. The L2 ganglia were consistent in location and became our most dependable landmark. There was some variability in location of other ganglia although this was not as prevalent as it seems to be in humans [3]. Bilateral denervation indicates that the great majority of postganglionic neurons rise in, or pass through, the L 3 - L 4 region. The remaining cells either originate caudal to L4 or leave the chain cranial to L3. Since denervation at the L4-L5 level was much less effective in reducing fluorescence than was L3-L4, it appears that many postganglionic axons exit the sympathetic chain in the area of L3. Bilateral removal of L2 suggests that 50% or more of postganglionic cell bodies originates at this level or cranial to it. Furthermore, bilateral removal of L2-L3 was nearly as effective as removal of L3-L4, indicating that not many postganglionic cell bodies originate caudal to L3. When comparing results of unilateral and bilateral denervation, the ipsilateral side of unilateral animals was not as dramatically changed as with bilateral denervation (Table I). For example, unilateral removal of L 3 - L 4 caused 59% reduction in ipsilateral fluorescence whereas on the contralateral side, there was a 17% average reduction. In contrast, bilateral removal of L 3 - L 4 caused 90% loss on both sides. This supports the possibility of a small amount of contralateral sympathetic innervation of postganglionic neurons to the hind-limbs. This is reinforced by previous observations in humans in which cross-communication was commonly observed especially in the 4th or 5th lumbar segments [9,10]. Additionally, recent studies in our laboratory using the horseradish peroxidase technique have demonstrated some postganglionic cell bodies in the contralateral sympathetic chain of the rat (Norvell and Peterson, unpublished results). In humans, preganglionic neurons do not enter the sympathetic chains below L2 or L3 [4]. Available anatomical drawings suggest that the rat may have white rami emerging as low as the S1 segment [7]. More recent histological studies, however, indicate that very few, if any, sympathetic preganglionic fibers emerge from the
174
spinal cord below L2 in the rat [2]. Thus, based on our findings that 90% of postganglionic cells pass through the L3-L4 area and the probability that if postganglionic cells originate below L4, they would be decentralized by cutting their associated preganglionic cells, the only intact pathways which may still be influencing the hind-limb must exit the chain above L3. Hence, though removal of L2-L3 was not as effective at reducing fluorescence as L3-L4, it may be more effective in decentralizing sympathetic innervation to the hind-legs. It is apparent that two-segment removal of the lumbar sympathetic chains cannot be used to eliminate postganglionic innervation of the hind-limbs. However, it is also apparent that bilateral removal of either the L2-L3 or L3-L4 segments will dramatically interfere with sympathetic control and yet permit minimal surgical trauma to the rat. The authors thank Lisa Flake and Jackie Rivera for their technical assistance. This work was supported by American Heart Association Grant 831047.
References 1 Christensen, N.J. and Galbo, H., Sympathetic nervous activity during exercise, Ann. Rev. Physiol., 45 (1983) 139-153. 2 Coggeshall, R.E., Emery, D.G., Ito, H. and Maynard, C.W., Unmyelinated and small myelinated axons in rat ventral roots, J. comp. Neurol., 173 (1977) 175-184. 3 Cowley, R.A. and Yeager, G.H., Anatomical observations on the lumbar sympathetic nervous system, Surgery, 25 (1949) 880-890. 4 Crosby, E.C., Humphrey, T. and Lauer, E.W., Correlative Anatomy of the Nervous System, MacMillan, New York, 1962, 522 pp. 5 De la Torte, J.C., An improved approach to histofhiorescence using the SPG method for tissue monoamines, J. Neurosci. Meth., 3 (1980) 1-5. 6 Donald, D.E., Rowlands, D.J. and Ferguson, D.A., Similarity of blood flow in the normal and the sympathectomized dog hind limb during graded exercise, Circulat. Res., 26 (1970) 185-199. 7 Greene, E.C., Anatomy of the Rat, Hafner, New York, 1963, 135 pp. 8 Karlsson, J. and Smith, H.J., The effect of lumbar sympathectomy on fiber composition, contractility of skeletal muscle and regulation of central circulation in dogs, Acta physiol scand., 119 (1983) 1-6. 9 Simeone, F.A., The lumbar sympathetic: anatomy and surgical implications, Acta chir. belg. 1 (1977) 17-26. 10 Webber, R.H., An analysis of the cross communications between the sympathetic trunks in the lumbar region in man, Ann. Surg., 145 (1957) 365-370.
171
Elsevier JAN 00440
Short Communications
Sympathetic postganglionic pathways to the hind-limb of the rat: a fluorescent histochemical study D. Fred Peterson and John E. Norvell Departments of Physiology and Anatomy, Oral Roberts UniversiO' Schools of Medicine and Dentistry, Tulsa, OK 74171 (U.S.A.)
(Received November 13th, 1984) (Revised version receivedJanuary 25th, 1985) (Accepted February 4th, 1985)
K e y words: sympathectomy - autonomic nerves - lumbar sympathetic chain
Our findings indicate that, in the rat, (1) 90% of sympathetic postganglionic fibers innervating the femoral arteries originate in or pass through the L 3 - L 4 sympathetic chain ganglia and (2) a small amount of contralateral innervation is likely in the lumbar region. Sympathetic innervation of resistance vessels in mammals contributes to distribution of blood flow in exercise [1]. In order to study sympathetic influences on blood flow in conscious animals, alteration of sympathetic activity must be accomplished without interfering with motor control of the exercising muscle. This has been accomplished in the dog by extensive lumbar sympathectomy [6]. Because of the small size of the rat and the possibility that surgical trauma would interfere with normal exercise, more limited sympathectomy, if possible, may be necessary in this species. The present study was designed to determine the course of sympathetic postganglionic fibers in the lumbar sympathetic chain supplying the femoral arteries. Its purpose was to identify the minimal denervation necessary to interrupt sympathetic outflow to the hind-limbs of the rat. Forty-three male Sprague-Dawley rats (350-500 g) underwent surgery to remove a section of the abdominal sympathetic trunk. Sham operations were performed on 5 additional rats under sodium pentobarbital anesthesia (30 m g / k g ) , a left abdominal incision was made above the kidney. The aorta was reflected ventrally and left psoas muscles reflected laterally thus exposing the sympathetic chains.
Correspondence: D.F. Peterson, Department of Physiology,Oral Roberts University School of Medicine, Tulsa, OK 74171, U.S.A.
0165-1838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
172 Sections of one or both chains and associated ganglia between T I 3 and L5 were then removed and the incision was sutured closed. Each rat was given a single dose of 30,000 units of penicillin (Bicillin, Wyeth) and allowed to recover between 6 - 1 7 days. The histofluorescence technique of De la Torre [5], utilizing glyoxylic acid was employed to determine the presence of catecholamines in the adventitia of the femoral artery and thus estimate the extent of interruption of postganglionic pathways to the hind-limb. Rats were decapitated and the pectineus muscle and associated femoral artery removed from both hind-legs. Heart muscle (atria) was also removed as a reference. The tissue was quick-frozen in 2-methylbutane cooled by liquid nitrogen and 20/~m sections were cut and treated. Sections were examined and photographed under fluorescence microscopy. If the atrial tissue did not indicate extensive presence of catecholamine, the animal was rejected. Sections containing the femoral artery were subjectively graded for fluorescence intensity on a scale of 0 - 3 . The grader did not k n o w the extent of the s y m p a t h e c t o m y at the time of grading. Zero indicated the absence of fluorescence and 3 indicated m a x i m u m intensity. Table I compares the intensity of fluorescence in the right and left femoral arteries to the extent of lumbar sympathectomy. It is apparent that when the left chain was removed from, and including the L 1 - L 5 ganglia that there was major loss of fluorescence in the left leg. In contrast, fluorescence in the right leg, did not indicate a significant reduction (Table I). W h e n L 3 - L 4 were removed on the left side, left femoral fluorescence was significantly reduced ( - 59%), but it was not as impressive as after L 1 - L 5 removals. Right side fluorescence tended to be reduced but it did not prove to be significant. Removal of T 1 3 - L 2 significantly reduced fluorescence on the left side but did not affect the right side.
TABLE I PRESENCE OF CATECHOLAMINE FLUORESCENCE IN THE FEMORAL ARTERY AFTER SELECTIVE REMOVAL OF SYMPATHETIC CHAINS Values represent averaged intensities (mean + S.E.M.) of fluorescence with 3 = maximum. B/S, sections of both chains removed; L, sections of left chain removed. Statistics performed were a one way analysis of variance and Duncan's multi-range test, Unbroken vertical bars indicate no significant difference exists between inclusive values. For example, under Left femoral, B/S L3-L4 is significantly different from both control and B/S L4-L5 but not significantly different from any other values. Control B/S L4-L5 L L3-L4 L T13-L2 B/S L2 B/S L2-L3 L L1-L5 B/S L3-L4
Left femoral
(n)
2.30 + 0.34 1.50+0.50 0.94 + 0.29 0.90+0.47 0.70 + 0.49 0.50-t-0.33 0.37+0.43 0.24+0.15
5 5 8
4 5 4 4 11
Control L T13-L2 L El-L5 L L3-L4 B/S IA-L5 B/S L2 B/S L2-L3 B/S L3-L4
Right femoral
(n)
2.80 + 0.14 [ 2.63 + 0.45 ] 2.38 + 0.301 2.33+0.291 1.40+0.45 ] 1.40 ± 0.45 0.80+0.52 I 0.27+0.08 [
5 4 4 9 5 5 5 11
173
The dramatic difference between ipsilateral and contralateral femoral fluorescence after unilateral denervation suggests strong dominance of ipsilateral sympathetic innervation to the hindqimb in rats. This has been assumed to be true in earlier studies on dogs [6,8]. On the other hand, since some fluorescence persisted on the ipsilateral side, our data suggest that there may be a small amount of cross-over from the contralateral lumbar sympathetic chain. The effect of bilateral removal of pairs of sympathetic chain ganglia was compared with unilateral removal (Table I). Bilateral removal of the L4-L5 ganglia caused 35-50% reduction in fluorescence. Bilateral removal of the L 3 - L 4 reduced fluorescence by 90% in both hind-legs, whereas removal of L 2 - L 3 reduced fluorescence by 71-78%. Finally, if the L2 ganglia were both removed, 50-70% reduction in fluorescence was observed. From a left side incision intended as a chronic surgical procedure in exercise studies, we found it technically difficult to locate and remove the right L1 ganglion without damaging the crural ligament. In fact, the right L2 ganglion is ordinarily not visible and is found by tracing the right sympathetic chain as it passes through folds of the ligament. Hence, we did not attempt bilateral denervation above the L2 level. The L2 ganglia were consistent in location and became our most dependable landmark. There was some variability in location of other ganglia although this was not as prevalent as it seems to be in humans [3]. Bilateral denervation indicates that the great majority of postganglionic neurons rise in, or pass through, the L 3 - L 4 region. The remaining cells either originate caudal to L4 or leave the chain cranial to L3. Since denervation at the L4-L5 level was much less effective in reducing fluorescence than was L3-L4, it appears that many postganglionic axons exit the sympathetic chain in the area of L3. Bilateral removal of L2 suggests that 50% or more of postganglionic cell bodies originates at this level or cranial to it. Furthermore, bilateral removal of L2-L3 was nearly as effective as removal of L3-L4, indicating that not many postganglionic cell bodies originate caudal to L3. When comparing results of unilateral and bilateral denervation, the ipsilateral side of unilateral animals was not as dramatically changed as with bilateral denervation (Table I). For example, unilateral removal of L 3 - L 4 caused 59% reduction in ipsilateral fluorescence whereas on the contralateral side, there was a 17% average reduction. In contrast, bilateral removal of L 3 - L 4 caused 90% loss on both sides. This supports the possibility of a small amount of contralateral sympathetic innervation of postganglionic neurons to the hind-limbs. This is reinforced by previous observations in humans in which cross-communication was commonly observed especially in the 4th or 5th lumbar segments [9,10]. Additionally, recent studies in our laboratory using the horseradish peroxidase technique have demonstrated some postganglionic cell bodies in the contralateral sympathetic chain of the rat (Norvell and Peterson, unpublished results). In humans, preganglionic neurons do not enter the sympathetic chains below L2 or L3 [4]. Available anatomical drawings suggest that the rat may have white rami emerging as low as the S1 segment [7]. More recent histological studies, however, indicate that very few, if any, sympathetic preganglionic fibers emerge from the
174
spinal cord below L2 in the rat [2]. Thus, based on our findings that 90% of postganglionic cells pass through the L3-L4 area and the probability that if postganglionic cells originate below L4, they would be decentralized by cutting their associated preganglionic cells, the only intact pathways which may still be influencing the hind-limb must exit the chain above L3. Hence, though removal of L2-L3 was not as effective at reducing fluorescence as L3-L4, it may be more effective in decentralizing sympathetic innervation to the hind-legs. It is apparent that two-segment removal of the lumbar sympathetic chains cannot be used to eliminate postganglionic innervation of the hind-limbs. However, it is also apparent that bilateral removal of either the L2-L3 or L3-L4 segments will dramatically interfere with sympathetic control and yet permit minimal surgical trauma to the rat. The authors thank Lisa Flake and Jackie Rivera for their technical assistance. This work was supported by American Heart Association Grant 831047.
References 1 Christensen, N.J. and Galbo, H., Sympathetic nervous activity during exercise, Ann. Rev. Physiol., 45 (1983) 139-153. 2 Coggeshall, R.E., Emery, D.G., Ito, H. and Maynard, C.W., Unmyelinated and small myelinated axons in rat ventral roots, J. comp. Neurol., 173 (1977) 175-184. 3 Cowley, R.A. and Yeager, G.H., Anatomical observations on the lumbar sympathetic nervous system, Surgery, 25 (1949) 880-890. 4 Crosby, E.C., Humphrey, T. and Lauer, E.W., Correlative Anatomy of the Nervous System, MacMillan, New York, 1962, 522 pp. 5 De la Torte, J.C., An improved approach to histofhiorescence using the SPG method for tissue monoamines, J. Neurosci. Meth., 3 (1980) 1-5. 6 Donald, D.E., Rowlands, D.J. and Ferguson, D.A., Similarity of blood flow in the normal and the sympathectomized dog hind limb during graded exercise, Circulat. Res., 26 (1970) 185-199. 7 Greene, E.C., Anatomy of the Rat, Hafner, New York, 1963, 135 pp. 8 Karlsson, J. and Smith, H.J., The effect of lumbar sympathectomy on fiber composition, contractility of skeletal muscle and regulation of central circulation in dogs, Acta physiol scand., 119 (1983) 1-6. 9 Simeone, F.A., The lumbar sympathetic: anatomy and surgical implications, Acta chir. belg. 1 (1977) 17-26. 10 Webber, R.H., An analysis of the cross communications between the sympathetic trunks in the lumbar region in man, Ann. Surg., 145 (1957) 365-370.