Decreased horseradish peroxidase labeling in deafferented spinal motoneurons of the rat

Brain Research, 275 (1983) 203--214 Elsevier

203

Research Reports

Decreased Horseradish Peroxidase Labeling in Deafferented Spinal Motoneurons of the Rat JEAN-MARIE PEYRONNARD and LOUISE CHARRON Centre de Recherche en Sciences Neurologiques and H~tel-Dieu Hospital, Universit~ de Montreal, Montreal H3C 3J7 QuL (Canada) (Accepted February 1st, 1983) Key words: horseradish peroxidase - - motoneurons - - rat - - deafferentation - - spinal cord - - retrograde transport

It has been suggested29,5°that the incorporation and retrograde transport of horseradish peroxidase (HRP) were linked to the level of neuronal activity. Therefore one could postulate that the motor impairment resulting from dorsal rhizotomy affects the HRP labeling of spinal motoneurons in the absence of morphological damage to the motor system. This hypothesis was tested in the adult rat by sectioning bilaterally the L3--L5 dorsal roots. 2-18 months after surgery, the L4 radicular nerve was immersed in a solution of HRP. Labeled motoneurons were counted together with the motor axons of the L4ventral root and results were compared with values obtained in paired controls. Deafferentation resulted in a crippling deficit of lower limb movements with disuse atrophy of muscle fibers but had no effect on the fiber population of the sciatic nerve and the L4 ventral root. Whereas in normal animals the L4 HRP-labeled motoneurons represented 71.9-98.3% (average 85.4) of the motor axonal counts, in animals studied 4, 12 and 18 months after dorsal rhizotomy, the number of motoneurons containing HRP granules constituted only 20.1-55.7% (average 46.2) of the number of motor axons and many of the labeled cells were faintly stained. These findings, which may reflect either a decreased retrograde transport of HRP in deafferented motoneurons or an increased turnover of the enzyme in the cell body, call attention to the possibility that the degree of activity in neuronal pathways influences HRP labeling. INTRODUCTION

MATERIALS AND METHODS

In adult m a m m a l s , deafferentation of a limb by dorsal rhizotomy produces movement deftcits 12,13,3°,4°,52,54,57, but has little d o c u m e n t e d effects

Surgical deafferentation The e x p e r i m e n t a l animals consisted of 4-monthold S p r a g u e - D a w l e y female rats. U n d e r general anesthesia induced with sodium p e n t o b a r b i t a l , a partial l a m i n e c t o m y was p e r f o r m e d in o r d e r to expose the L3, L4 and L 5 dorsal roots bilaterally. The roots were cut proximally to the spinal ganglia and a 1-mm-long segment was resected in an a t t e m p t to prevent regeneration. U p o n recovery, each rat was k e p t with an u n o p e r a t e d control animal of the same age, in a softly p a d d e d plastic cage. Periodic examinations allowed p r o m p t t r e a t m e n t of m i n o r injuries and consequently, none of the d e a f f e r e n t e d animals r e p o r t e d in the present series d e v e l o p e d significant soft tissue lesions of the hind limbs.

on the m o r p h o l o g i c a l or electrophysiological p r o p erties of the spinal m o t o r cells and their axons4.6, 27. In the visual system, however, it has been suggested 50 that sensory deprivation m a y affect certain neuronal functions such as the ability to i n c o r p o r a t e and retrogradely transport the horseradish peroxidase ( H R P ) . To further evaluate this possibility, the L4 m o t o n e u rons of the rat were d e a f f e r e n t e d by sectioning bilaterally the L a - L 5 dorsal roots. A t various times after the o p e r a t i o n , the L4 m o t o n e u r o n s were l a b e l e d with H R P and c o u n t e d t o g e t h e r with the m o t o r axons in the corresponding ventral roots. Results were comp a r e d with values o b t a i n e d in p a i r e d controls.

0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.

204

Horseradish peroxidase labeling 2, 4, 12 and 18 months after the operation, deafferented animals and their controls were anesthetized. The right L 4 spinal nerve was transected and its proximal stump immersed for 3 h in a capillary tube filled with a 20% solution of H R P (Sigma, type VI). After a survival period of 24--48 h, the animals were reanesthetized and the right tibialis anterior (TA) muscle was dissected and frozen in liquid nitrogen for further histological studies. Subsequently, the animals were perfused through the heart with a solution made of 1% paraformaldehyde and 1.25% glutaraldehyde in phosphate buffer at pH 7.4.

Histological processing (a) Spinal cord. The L3-L 5 spinal cord segments were removed and frozen in a single block. Serial 20-/~m-thick sections were treated with tetramethyl-

benzidine 36,37 and counterstained with neutral red. After a microscopic examination under bright- and dark-field illumination at 400 x magnification, cells containing HRP granules were plotted on photographic prints (x 50) of the spinal cord sections. These prints were superimposed to reconstruct and analyze quantitatively the motor cell columns. In this process, consideration was given to the fact that lumbospinal motoneurons of the rat have diameters ranging from 15 to 70/~m 42. Consequently, in our 20/~m-thick preparations, cell contours visualized in a single section were considered indicative of the presence of a small neuron, when nuclei or nucleoli were poorly distinguishable. In the case of cellular profiles repeating in the same location on several consecutive sections, three-dimensional cell reconstruction was obtained through measurements with an eyepiece micrometer of mean cellular diameters 5, and helped

Fig. 1. Photographs of deafferented rats showing postural abnormalities of lower limbs at rest (A), on vertical displacement (B) and during locomotion (C and D).

205

TABLE I Myelinated fiber densities and caliber spectra in sciatic nerves Months Alter Surgery

2 4 12 18

Controls

Deafferented animals MF density (per sq. ram) mean + S.D.

Percentage of fibers > 8am mean + S.D.

MF density (per sq. ram) mean + S.D.

Percentage of fibers > 81~m mean + S.D.

15,176 + 265 14,801 + 159 15,213 + 237 15,261 + 418

40.6 + 4.2 38.9 + 1.0 37.7 + 1.7 41.2 + 3.2

15,139 + 442 14,908 + 239 15,121 _+445 14,983 + 133

39.6 + 37.8 + 42.2 + 37.6 +

1.8 3.7 2.7 2.2

dorsal roots in order to evaluate the completeness of

to distinguish between two different neurons. (b) Nerves and muscles. The right L4 ventral roots

were dissected for quantitative analysis of the motor axonal population. In deafferented animals, additional samplings were taken of the atrophic lumbar

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deafferentation. In these animals, the L3-L 5 spinal nerves and the sciatics were also examined, searching for morphological evidence of peripheral nerve degeneration or regeneration, which could have re-

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Fig. 2. Left: cross-section of part of the sciatic nerves with no evidence of fiber damage in the deafferented animal (top, DT). Control (C7) in bottom. × 240. Right: myelinated fiber size histograms are similar in the deafferented (top) and control animals (bottom).

206 suited from motor fiber damage during the dorsal rhizotomy. All nerve specimens were postfixed in 2% osmic acid and processed for epoxy-resin embedding. Photographs of semithin transverse sections, enlarged to a final magnification of 1000 x were used for the analysis of myelinated fiber (MF) density and caliber with the help of a particle size analyzer. The right TA muscles were cut transversely in a freezing microtome and the 15-gm-thick sections were reacted with the myofibrillar adenosine triphosphatase following preincubation at pH 4.5. Number and diameters of muscle fibers and the respective ratios of the various fiber types were determined on photographic enlargements (x 200) of the TA sections.

RESULTS Clinical observation Immediately following the dorsal rhizotomy, the animals recovered a quadrupedal locomotion and did not exhibit the transient flaccid paralysis described in cats 13,14. However, they showed obvious and persistent motor handicaps. At rest, they usually kept their hindlimbs hyperextended and outstretched to the side (Fig. 1A). Vertical displacement or holding of the animal by the tail induced a sustained extensor response of the thighs and legs with a plantar flexion of the feet and toes. In some instances this posture was interrupted or replaced by forceful flexion of the hips and knees (Fig. 1B). The gait was usually awkward with overabduction of the hindlimbs (Fig. 1C) and increased tilting of the body. It was occasionally delayed by a prolonged extension of one limb which

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Fig. 3. Left: cross-section of the inner part of tibialis anterior muscles showing in the operated animal (top D9) a mosaic distribution of myofibers comparable to control (bottom C9). × 45. Right: diameter histograms document the hypotrophy of the myofibers in the deafferented rat (top) as compared to control (bottom).

207 TABLE II Number of myofibers and percentage of respectivefiber types in TA muscles Months after surgery

2 4 12 18

Deafferented animals

Controls

Mean (± S.D.) number of myofibers

Mean(± S.D.) percentage of fiber types

Mean (± S.D.) number of myofibers

1

2A

2B

14,570±4~ 13,891±5~ 15,454±717 14,973±355

2.9±0.6 3.0±0.3 3.2±0.8 2.6±0.4

19.5±1.3 21.0±0.7 20.0±2.0 20.9±1.1

77.6±1.2 76.0±0.7 76.8±1.3 76.3±0.7

made floor contact with the dorsum of the foot (Fig. 1D). Treadmill walking revealed a dissociation between the well-coordinated movements of the forelimbs and the slow and inaccurate displacements of the hindlimbs which at times were simply kept immobile. Histological evaluation of peripheral nerves and TA muscles in deafferented animals Several animals were excluded from the study as the presence of damaged axons or small regenerating fibers in the sciatic nerves was traced to a lesion of a lumbar ventral root which had probably been inflicted at the time of surgery. In the remaining 12 animals, dorsal rhizotomy had no morphological effect on peripheral nerve fibers, MF densities and caliber spectra of the sciatic nerves being comparable in operated and control animals (Table I and Fig. 2). His00 E .~

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14,~7±7~ 14,735±358 13,915±560 14,863±683

Mean(± S.D.) percentage of fiber types 1

2A

2B

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20.9±2.0 21.2±1.3 21.7±1.6 20.4±1.1

76.5±2.4 76.7±0.7 75.6±1.3 76.6±1.1

tochemical studies of the TA innervated mainly by the L4 root 44 revealed that myofibers had retained their normal mosaic distribution (Fig. 3) and were only reduced in size, in keeping with the conspicuous hypotrophy of the hindlimb muscles. Quantitative analysis (Figs. 3 and 4) showed this volumetric reduction to affect all fiber types with no change in the overall composition of the TA, the respective percentages of type I, 2A and 2B fibers being similar in deafferented and control animals (Table II). Histological assessment of the lumbar dorsal roots following rhizotomy Histological studies confirmed that, proximal to the site of section, dorsal roots consisted of a fibrous tissue devoided of nerve fibers (Fig. 5A). Occasionally, in a long-term animal, partial re-innervation of the proximal radicular stump had occurred as indicated by the presence of small regenerating myelinated fibers (Fig. 5B). Comparative analysis of L~ motor axonal and motoneuronal populations in normal and deafferented animals As shown in Table III, with individual variations not related to the age of the animal, the numbers of MF in the L4 ventral roots of normal rats (range 1579-2229, average 1891) were comparable to values obtained after deafferentation (range 1469-2575, average 1958). As in the example of Fig. 6, the fiber size histograms were in all cases clearly bimodal, the average percentage of large (> 8/am) and small (< 8/~m) MF being, respectively, 59 and 41% in normal rats, and 56 and 44% in operated animals. Comparison in normal animals between counts of motor axons and HRP-labeled motoneurons gives

208

T A B L E III

Comparison between counts of motor axons and HRP-labeled motoneurons Control animals MF

HRP

Months after surgery

Deafferented animals

HRP (%)

MF

HRP

MF C1 C2 C3

1579 1909 1903

1155 1812 1622

73.1 94.9 85.2

Mean

1797

1529

84.4

C4 C5 C6

1779 2016 1790

1749 1863 1503

98.3 92.4 83.9

Mean

1861

1705

91.5

C7 Cs C9

2221 2229 1815

1773 1629 1772

79.8 73.0 97.6

Mean

2088

1724

83.4

Clo Cll C12

1834 1615 2012

1319 1370 1813

71.9 84.8 90.1

Mean

1820

1501

82.2

HRP (%) MF

2 2 2

4 4 4

12 12 12

18 18 18

Dl D~ D3

D4 D5 D6

D7 Ds D9

Dlo DI1 Dl2

1643 1731 1928

1321 1526 1407

80.4 88.1 72.9

1767

1418

80.4

1800 2236 2050

1004 1107 1095

55.7 49.5 53.4

2029

1069

52.8

1469 1775 2492

819 560 1055

55.7 31.5 42.3

1912

811

43.1

1995 2575 1803

1101 520 964

55.1 20.1 53.4

2124

861

42.8

Fig, 5. A: cross-section of the L 4 dorsal root with complete absence of myelinated fibers (animal D9). × 380. B: partial reinnervation of the L 5 dorsal root 18 m o n t h s after surgery (animal D12). × 380.

209 spective of the delay between the administration of HRP and the time of sacrifice. The length of the L 4 motor cell column (Fig. 8) being similar for normal and deafferented animals suggested that the decreased neuronal labeling associated with the dorsal rhizotomy affected neurons over the entire L 4 spinal cord segment and was not confined to certain areas.

an idea of the accuracy to be expected with the HRP technique when assessing quantitatively a large cell population such as the L4 motoneurons. Data summarized in Table III indicate that HRP estimates were to variable degrees lower than axonal counts as they represented between 71.9 and 98.3% (average 85.4%) of the numbers of myelinated fibers in the L 4 ventral roots. These differences were significantly amplified in long-term deafferented animals (Table III). Whereas in rats studied 2 months after dorsal rhizotomy the L 4 motoneuronal population labeled with HRP did not differ from controls, studies performed in animals deafferented 4, 12 and 18 months earlier indicated abnormalities of neuronal labeling. In this group, not only were the numbers of motoneurons containing HRP granules reduced to the point that they constituted only 20.1-55.7% (average 46.2%) of the number of motor axons, but also many of the labeled cells were faintly stained (Fig. 7) irre-

DISCUSSION Processes by which nerve cells respond to alterations of the afferent inputs have been extensively reviewedTA2,46. Results vary according to species, age and experimental procedure, removal of presynaptic structures producing, for instance, more obvious morphological changes than functional disruption of the afferent traffic12. Certain systems also appear more susceptible than others to the effect of anterograde transneuronal degeneration. In this respect,

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210

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Fig. 7. Cross-sections of anterior horns ( x 40) taken 500 am apart and closer views of representative motoneurons ( x 280) showing intense HRP-labeling in control animals (left) and faintly stained neurons in deafferented rats (right).

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the well-documented degenerative changes induced by sensory deprivation in the lateral geniculate neurons of young 19 and adult animals6,31.32, contrast with the conflicting reports on the effect of deafferentation on spinal cord motoneurons in mammals. Chromatolysis of anterior horn cells was noted following section of dorsal roots at thoracic levels in cat 55 or in the lumbar region in man and monkey 11. However, this phenomenon was considered insignificant in another study dealing with cats killed 3-6 months after unilateral severance of L6-S 1 dorsal roots and ablation of the motor cortex 6. In the rat, it has been stated, but not documented, that severe degeneration of ventral horn cells could be induced by dorsal rhizotomy, especially when it was combined with cord transection 2. However, in puppies, lumbosacral motoneurons isolated by cord section on either side of deafferented roots 53 were capable of surviving intact. More recently, histochemical changes together with a decrease in size and number of spinal cord motoneurons were said to take place within a week not only in the isolated spinal cord preparation but also after a dorsal rhizotomy in adult cats 58. On the contrary, other authors have concluded that deafferentation did not change the diameter and conduction velocity of motor axons in mature cats 4,27 and had only a limited effect in growing rats 48. Various reasons may explain such conflicting reports. Positive evidence is often based on neuronal alterations of

disputable significance or on variations in nerve cell populations difficult to appreciate with conventional histological techniques. On the other hand, negative morphological evidence is of limited value, since, as previously stated 33, the normal appearance of a neuron does not imply normal function. Because horseradish peroxidase is a physiological marker 3s which requires active uptake by nerve terminals and retrograde axonal transport, these processes being eventually influenced by the extent of neuronal activity29, 50, we used this enzyme in order first to determine the number of motoneurons in the L 4 spinal segment of normal rats, and second to appreciate differences in the labeling of L 4 n e u r o n s following lumbodorsal rhizotomy. Despite the recent availability of well-standardized H R P histochemical techniques 36,37,47 applying to the mapping of lower motoneurons 1,3,5,9,16,17,18,20,23,24,26,28,34,45, accuracy of the results obtained has rarely been tested. A comparison made between counts of labeled motor cells to soleus and gastrocnemius of cats 5, and numbers of motor fibers previously reported 4 in deafferented nerves to these muscles, revealed a marked deficit of small motoneurons. One explanation for this discrepancy being a limited access of H R P injected intramuscularly to encapsulated terminals of gamma-motor fibers 45, some authors were led to prefer the bathing of proximal stumps of cut nerves in a solution of HRp3,43, 45,51,56 in order to expose axons to a uniform concentration of the enzyme. This method applied to the analysis of the innervation of the rat sternomastoid muscle 17 revealed a good correlation between histograms of motoneurons and motor axons, the latter being differentiated from sensory fibers by acetylcholinesterase histochemistry. In the present study, the numbers of HRP-labeled motoneurons in the L 4 spinal segments of normal rats were found to be somewhat lower than the numbers of motor axons in the corresponding ventral roots. The ratios neuronal counts/axonal counts expressed in percentage varied from 71.9 to 98.3% (average 85.4) in young and aged animals with no indication of a relationship with the degenerative process reported in the spinal cord of old rats 4t. Rather than being explained by branching of ventral root axons, these differences probably reflect a slight inaccuracy of the motoneuronal estimates, hardly surprising if one considers the difficulty of identifying all labeled structures in a pool

212 averaging 1891 cells and likely to contain, in view of the composition of the ventral roots, over 40% of small neurons not easy to detect. Deafferentation had no appreciable morphological effects on the motor axons, the population and caliber spectrum of MF in the L 4 ventral roots being identical in control and operated animals up to 18 months after surgery. However, the percentage of HRP-labeled motoneurons, which was comparable to controls in the animals deafferented 2 months earlier, decreased significantly in rats tested 4, 12 and 18 months after the operation, the average percentages being respectively 52.8, 43.1 and 42.8 with individual values as low as 20.1%. It is unlikely that these resuits reflect neuronal retrograde changes due to a possible injury of the motor axons at a site distal to the level of HRP administration and root examination. Indeed, despite the fact that peripheral nerves may be compressed and damaged through the disability produced by deafferenting the hindlimb, there was no histological evidence of damage to the sciatic nerves in any of the animals included in this study. Furthermore, histochemical examination of the tibialis anterior muscles receiving most of their motor supply from the L4 root 44 revealed only a mild to moderate atrophy of the myofibers related to disuse 8, and no fiber type grouping as it occurs after partial denervation22,25, 44. Thus, it is reasonable to assume that our findings are a consequence of the dorsal rhizotomy, probably on the basis of functional changes affecting the HRP turnover in deafferented motoneurons. The effectiveness of deafferentation or other procedures such as tenotomy for reducing the input activity in motor nerves is controversial49 but it is generally accepted that one or the other method limits the motor traffic 1°,21,48. Moreover, several studies in monkey 30,4°,52,5<57 and cat 13,14,15.35 have documented the motor impairment caused by dorsal rhizotomy and the various mechanisms of recovery involved, such as the unmasking of other afferent path-

REFERENCES 1 Akagi, Y., The localization of the motor neurons innervating the extraocular muscles in the oculomotor nuclei of the cat and rabbit, using horseradish peroxidase, J. comp. Neurol., 181 (1978) 745-762. 2 Barron, D. H., Structural changes in anterior horn cells following central lesions. In Proceedings of the Society for Ex-

ways, the sprouting of adjacent dorsal roots and descending intraspinal projections, changes in the responsiveness of spinal cord cells to which one may add attempted regeneration of severed dorsal roots as we observed occasionally. Judging from clinical observations, none of these mechanisms was sufficient to restore normal posture and walking in the deafferented rats which exhibited a crippling hindlimb dysfunction up to 18 months after the lumbodorsal rhizotomy. In conclusion, deprivation of peripheral sensory inputs seems to have long-lasting functional effects on rat spinal cord motoneurons including the fact that within 4 months, nearly half of the cells appear unable to accumulate in their perikarya an amount of HRP sufficient to be detected by light microscopic examination. This observation adds weight to the hypothesis that the degree of activity in neural pathways influences HRP labeling 5° as suggested by previous reports of a decreased peroxidase staining in lateral geniculate neurons of cats after enucleation 50 and of an increased uptake of HRP in the electrically stimulated frog sartorius nerve 29. The biological basis for this phenomenon remains unclear but implies additional mechanisms to the changes in the amount of HRP picked up by nerve terminals29, 50, since in our study the enzyme was delivered directly to the axons. Therefore the reduced quantity of tracer found in the soma of deafferented motoneurons may reflect either a decreased axonal retrograde transport of H R P or an increased turnover of the enzyme in the cell body. ACKNOWLEDGEMENTS This work was supported by grants from the Medical Research Council of Canada (J.M.P.) and the Jeanne-Mance Foundation (L.C.). The assistance of Mrs. J. Perreault and C. Laurier is acknowledged with gratitude.

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