Influence of an experimental hindlimb maldevelopment on axon number and nodal spacing in the rat sciatic nerve. C. Hildebrand, M. Westerberg and G.Y. ...
Developmental Brain Research, 50 (1989) 169-175
169
Elsevier BRESD5~78
Influence of an experimental hindlimb maldevelopment on axon number and nodal spacing in the rat sciatic nerve C. Hildebrand, M. Westerberg and G.Y. Mustafa* Department of Cell Biology, Facultyof Health Sciences, University of Link6ping, Link6ping (Sweden) (Accepted 23 May 1989)
Key words: Rat sciatic nerve; Development; Axon number; Internodal length; Limb growth
In neonatal rat pups the femoral and tibial epiphyseal cartilages on the left side were coagulated with a microcautery device. The subsequent femoral and tibial growth in length was markedly restricted on the left side, but the foot and the pelvic region exhibited normal longitudinal growth. After 6 months the sciatic nerves were removed from both sides. Electron microscopic analysis of nerve specimens from the stunted side revealed that the number of axons was 20% less compared to control specimens. Light microscopic examination of teased preparations showed a normal nodal spacing in the pelvic segment but abnormally short internodes in the femoral segment of the left sciatic nerve. These results suggest that the number of axons in the rat sciatic nerve adapts to a target maldevelopment that sets in neonatally, and that internodal elongation during development proceeds according to the local growth in length of the nerve rather than to the length growth of the whole nerve.
INTRODUCTION D u r i n g early d e v e l o p m e n t , the outgrowth of axons to target areas involves an excess of neurons. T h o s e n e u r o n s which succeed in establishing peripheral contacts survive, and those which fail to do so die. In this way a large p r o p o r t i o n of the neurons originally p r e s e n t are e l i m i n a t e d 1'13'24'29. Experim e n t a l expansion of the target a r e a in i m m a t u r e animals m a y lead to survival of a larger p r o p o r t i o n of the n e u r o n p o p u l a t i o n and vice versa 29. A c c o r d ing to some workers, a second regressive event - elimination of s u p e r n u m e r a r y axonal branches - m a y occur at a later stage o f d e v e l o p m e n t 2°. These events relate the n u m b e r of axons in a p e r i p h e r a l nerve to the size of its target. In addition, the m y e l i n a t e d axons in a p e r i p h e r a l nerve are influe n c e d by d e v e l o p m e n t a l changes in the distance to the target. N o d a l spacing is greater in large than in
small m a t u r e m y e l i n a t e d p e r i p h e r a l nerve fibers, since internodes of large fibers are at h a n d during a longer p e r i o d of the animals growth in size, than those of small fibers. O n this basis, a regular positive relation b e t w e e n i n t e r n o d a l length and fiber diameter is established during d e v e l o p m e n t 12'21'28. In the rat sciatic nerve, internodal elongation matches the length growth during n o r m a l d e v e l o p m e n t (about 8 times) 17'1s'28, assuming that the initial internodal length is about 150 g m . In some developing nerves, e.g. the spinal roots 3'4'26, and the inferior alveolar nerve 1°'11 of the cat, the longitudinal relations are m o r e complex. In these nerves a mismatch b e t w e e n nerve growth and i n t e r n o d a l elongation elicits a d e v e l o p m e n t a l n o d a l - i n t e r n o d a l remodelling: elimination of some internodes allows the remaining ones to elongate m o r e than the nerve. In the present p a p e r we examine axon n u m b e r and i n t e r n o d a l lengths in the rat sciatic nerve, following an exper-
* Permanent address: Deans Office, Medical College, Tikrit University, P.O. Box 45, Tikrit, Iraq.
Correspondence: C. Hildebrand, Department of Cell Biology, Faculty of Health Sciences, University of Link6ping, S-581 85 Link6ping, Sweden. 0165-3806/89/$03.50 ~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
170 imental disturbance of the hindlimb's postnatal growth in length. Some of the results have been presented in abstract form 16. MATERIAL AND METHODS In order to identify the femoral and tibial epiphyseal plates, neonatal Wistar rat pups (Alab, Sweden) were fixed and stained with Alizarin. These pups were killed with ether, fixed in 70% ethanol (7 days), cleared in freshly prepared 1% K O H (4 days) and stained in a solution of 30 mg Alizarin red in 1000 ml 1% K O H (2 days). The stained preparations were then immersed in a mixture of 1 part benzyl alcohol, 2 parts 87% glycerol and 2 parts 70% ethanol (3 days) and stored in a 1:1 mixture of glycerol and 70% ethanol. Fig. 1 illustrates the hindlimbs in a stained rat pup. For surgery, 1 to 2-day-old rat pups were anesthetized by hypothermia. The femoral and tibial diaphyses were exposed on the left side through lateral and anteromedial skin incisions respectively, and blunt dissection, ascertaining that the sciatic nerve was unharmed. The sharpened tip (0.2 × 0.4 x 2.0 ram) of a microcautery device (Weller Temtronic soldering station, Cooper industries, Besigheim, E R . G . ) was inserted in the femoral and tibial epiphyseal cartilages. With the adjustable heat set at 175 °C, the tip was pushed into (but not beyond) each epiphyseal plate, and was allowed to remain there about 5 s, when the cartilage around the tip became opaque. Great care was taken
to avoid heat injury as well as mechanical injury to the surrounding soft tissues, particularly the sciatic nerve. The skin was sutured and the pups were returned to their mothers. Figs. 2 and 3 show a photograph and an X-ray picture respectively, from adult rats operated on as neonates. After 6 months survival, 3 operated rats were anesthetized with chloral hydrate, and perfused through the heart with a solution of 5% glutaraldehyde in a 300 mOsm phosphate buffer, also containing 0.1 M sucrose. The left (experimental) and right (control) sciatic nerves were removed, postfixed in glutaraldehyde, rinsed in buffer, osmicated and rinsed. From each nerve a short segment was taken from the level of the hamstring branch. This specimen was dehydrated with acetone and embedded in Vestopal W. Transverse thin sections were cut with an LKB Ultrotome III and collected on one hole copper grids coated with carbon-stabilized formvar. Following contrasting with uranyl acetate and lead citrate, the thin sections were examined in a J E O L JEM 2000 electron microscope. The number of myelinated axons was counted on montages of electron micrographs covering the entire nerve (1000x). The proportion of unmyelinated axons was obtained through sample countings (sample size 2500-3000 axons) and used to estimate the total number of unmyelinated axons. The pelvic and femoral sciatic nerve segments, above and below the hamstring branch level were kept overnight in 50% glycerin and were then stored in pure glycerin. The pieces were teased with needles and mounted in glycerin. Internodal lengths (L) and fiber diameters (D) were measured in the light microscope with the aid of a MOP Videoplan equipment. D was taken as the average of five separate measurements along each internode. The number of internodes measured per nerve varied around 120. Graphics and regression analysis were conducted in a Macintosh SE computer. RESULTS
4. Fig. 1. Alizarin-stained preparation of newborn rat pup,
showing epiphyseal plates of the hindlimbs.
In the operated rats a deficient left hindlimb growth was noticeable from one week after surgery and on. Following survival times exceeding one month, the thigh and leg were markedly shorter on the operated side than on the control side. Measure-
171
Fig. 2. Six-month-old rat with a growth-deficient left hindlimb. This rat was operated as a neonate.
Fig. 3. This X-ray picture shows a neonatally operated 6-month-old rat with a growth-deficient hindlimb on the left side. Note the exceptionally short femur and tibia.
ments on X-ray pictures revealed that the average combined femoral and tibial length in the growthdeficient limb was 40% of the control length (range 38-42%, n = 4). On the left side the pelvis and the foot were normally sized and had normal configurations, apart from an acetabular maldevelopment (Figs. 2 and 3). During dissection after perfusion it was apparent that the thigh and leg muscles on the left side were short and thick. The sciatic nerve trunk below the hamstring branch was extremely short and this resulted in early branching. Above the hamstring branch the sciatic nerve trunk had a normal length and general configuration. Electron microscopic analysis of cross-sections from sciatic nerve specimens showed clearcut left-
TABLE I Number of myelinated axons (MYEL AX), proportion of unmyelinated axon profiles (% UAP) and calculated total number of unmyelinated axon profiles (NO UAP) at the level of the hamstring branch in the sciatic nerves from growth-deficient (left) and control (righO sides Rat
MYEL A X
% UAP
NO UAP
no.
Left
1 2 3
Right Ratio Left Right Left l/r
Right Ratio l/r
6910 8772 0.79 69.9 60.5 11747 14035 0.84 7612 9353 0.81 60.2 66.0 12179 15526 0.78 8192 9624 0.85 63.8 63.5 13435 15687 0.86
Mean 7571 9250 0.82 64.6 63.3 12453 15083 0.83
right differences in the operated animals. On the stunted side the number of myelinated axons was about 82% of the number on the control side (Table I). The estimated proportion of unmyelinated axons was similar on the two sides, and the mean calculated total number of unmyelinated axons on the stunted side was 83% of the control value. Although the size range was bilaterally similar, the size distribution of the myelinated axons appeared to be shifted towards smaller sizes on the stunted side. Light microscopic examination of teased specimens from pelvic and femoral sciatic nerve levels in operated rats, revealed similar left-right differences in the 3 animals. On the control side, L increased from about 200 to some 1400/~m, as D increased from 2 to about 15/zm (Fig. 4). In the pelvic nerve segment on the stunted side, D varied from 2 to some 15/zm and the L values were about 200 p m in the lower fiber size range and some 1300/zm in the upper fiber size range, just as in the controls (Fig. 4). Hence, the inclinations of the regression lines describing the relation L / D in the pelvic sciatic nerve segments on the operated side, correspond to those found on the control side. In contrast, the femoral segment of the sciatic nerve on the operated side exhibits a clearly abnormal relation between L and D. While D ranged from 2-3 to about 15/zm, L varied around 200-300/~m in the lower fiber size range and some 600-800/zm in the upper size range (Fig. 4).
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173 DISCUSSION
The normal rat sciatic nerve contains about 8200 myelinated and some 15,500 unmyelinated axons at a level just distal to the sciatic tuberosity 19. This agrees reasonably well with our findings on the control side. The somewhat higher number found by us may be related to the fact that our counts include the hamstring nerve. The internodal lengths recorded on the control side similarly conform with previous observations ~7'18. On the stunted side, the number of myelinated axons was found to be subnormal in all cases, the average being some 80% of the number on the control side. Since the proportion of unmyelinated axons was largely similar on the two sides, the actual number of unmyelinated axons on the experimetal side was reduced in proportion to the reduction in number of myelinated axons. Hence, it appears that the total population of sciatic nerve axons was reduced to about 80% of the normal level in response to the experimental maldevelopment. Peripheral nerves contain a surplus of axons at early stages of development, before target contact has been established. This is followed by death and elimination of those neurons, which fail to establish target contact. In this way the number of sensory and motor neurons is adapted to the size of the target 1"13'24'29. The present observations suggest that the number of axons in the rat sciatic nerve adapts to a hindlimb maldevelopment that sets in neonatally. A postnatal loss of axons in the normal developing rat sciatic nerve has been reported by some workers 2°. The loss of sciatic axons observed here could either be due to a postnatal elimination of entire neurons in response to deficient target growth, or to an elimination of supernumerary axonal branches with preservation of the number of neurons projecting to the sciatic nerve 2°. The pattern of nodal spacings presently observed on the control side is in good agreement with previous results 17'18. As shown here, teased fiber
preparations from the pelvic segment of the sciatic nerve on the maldeveloped side showed a normal nodal spacing. In contrast, the same axons exhibit abnormally short internodes in the femoral segment of the sciatic nerve. Apparently, the nodal spacing was only reduced in sciatic nerve segments directly affected by the growth abnormality: longitudinal adjustments producing a uniform nodal spacing along the nerve did not occur. This suggests that the length growth of a specific internode proceeds according to the local growth in length in the immediate neighbourhood of that internode, rather than to the growth of the entire nerve. In the femoral nerve segment on the stunted side, the maximal L varied around 700 a m - - a deficit of about 50% compared to the controls. This corresponds approximately to the growth deficit exhibited by the long bones of the operated hindlimb. Thus, it seems that the internodes have elongated in accordance with the little growth of the long hindlimb bones that occurred in spite of the surgical intervention. We found little or no evidence for ongoing myelin sheath remodelling in teased nerve preparations from operated animals with survival times of 1, 3 and 6 months. Our observations indicate that the Schwann cells affected by the growth deficiency were incapable of a fully compensatory myelin sheath remodelling like that seen in some developing nerves 4'1°'11'26. It might be argued that the occurrence of short myelin sheaths in the femoral segment but not in the pelvic segment could be explained by unintentional nerve injury during surgery, and that we have examined regenerated axons. However, the relation between L and D in the femoral segment of the nerve in the growth deficient hindlimb was clearly different from the relation seen after regeneration following a neonatal nerve injury TM. For this reason (and since we were careful at surgery) we believe that the sciatic nerve alterations observed here are mainly due to the deficient growth. This brings up the issue of a differentiated
Fig. 4. Graphs on the left side describe the relation between internodai length (L) and fiber diameter (D) in the pelvic (filled dots) and femoral (empty circles) segments of the left (experimental) sciatic nerve in 3 rats (A, B and C), which were operated as neonates and survived 6 months. Graphs on the right side illustrate the relation L/D in the pelvic segment of the sciatic nerves on the normal control side of the same animals. Note that the inclinations of the linear regression lines for the relation L/D in the pelvic segments of the growth-deficient nerves are very similar to the controls whereas the relation is dearly abnormal in the femoral segment of the same nerve. Correlation coefficients varied around 0.90 (range 0.89-0.91) for the control nerves and the pelvic segment of the growth deficient nerves and around 0.79 (range 0.73-0.85) for the femoral segment of the growth-deficient nerves.
174 longitudinal growth in axons. According to studies in vitro, an axonal branch point formed through splitting of one growth cone into two can remain fixed relative to the cell body, while new axon segments appear between the branch point and the growth cones 7. Whether neurites elongate primarily because of 'pull' exerted by the growth cone or because of 'push' provided by anterograde transport and assembly of materials at the neurite tip has been discussed. It was recently shown that 'push' alone can support neurite elongation in vitro, and that 'pull' is unnecessary 2s. In young kittens, some unmyelinated motoraxon collateral segments elongate, whereas other segments of the same axons decrease in length during development 27. An example of a spatially differentiated longitudinal growth in myelinated axons is provided by the fact that the mechanically weakest segments - - the nodes of Ranvier - - do not grow in length during development 2A4. In contrast, the mechanically most resistant segments - - the internodes - - tend to elongate some 2-10 times during development (depending on the nerve and on whether the axon myelinates early or late). Another example of uneven growth is provided by spinal roots, where internodes adjacent to the PNS/CNS transition tend to be shorter than conventional internodes s. At least in some cases, myelinated axons may have unusually short internodes in direct relation to branch points 9. In myelinated cultures of dorsal root ganglia internode lengths vary between 54 and 246 /tm (mean 128 /~m)6. These workers concluded that L does not change appreciably once the cultures are fully myelinated. They interpreted the observation to confirm the importance of 'stretching' due to growth of surrounding tissue in the determination of internodal length. At least with respect to the in situ situation, however, there is evidence suggesting active growth in length of the internodes. In normal adult mouse sympathetic REFERENCES 1 Aguayo, A., Peyronnard, J.M., Terry, L.C., Romine, J.S. and Bray, G.M., Neonatal neuronal loss in rat superior cervical ganglia: retrograde effects on developing preganglionic axons and Schwann cells, J. Neurocytol., 5 (1976) 137-155. 2 Berthold, C.-H., Ultrastructure of postnatally developing feline peripheral nodes of Ranvier, Acta Soc. Med. Upsal., 73 (1968) 145-168.
ganglia some Schwann cells elongate very actively at the expense of their neighbors, although the axon does not grow in length zz'e3. In regenerated adult rat sciatic nerve axons, some internodes along the non-growing axons increase in length, while other internodes shorten and eventually become eliminated 15'w. To what extent this pattern, with alternating regions of internodal elongation and shortening, also involves the axolemma is unknown. In regenerating sciatic nerves of developing rats, it seems that the minimum internodal length increase needed amounts to 2x ~s. In the present situation, the length growth that occurred on the growth deficient side seems to have reached that minimum, so that myelin sheath remodelling was avoided. We propose that the internode represents a basic unit for length growth in myelinated axons, and that different internodes along a single axon do not necessarily behave similarly. In conclusion, the results of the present paper indicate that the number of axons in the rat sciatic nerve adapts to a target maldevelopment that sets in neonatally and that the internodal elongation during development proceeds according to the local growth in length of the nerve rather than to the length growth of the whole nerve. ACKNOWLEDGEMENTS
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