T H E ANATOMICAL RECORD 2 1 5 7 - 8 1 0986) Fiber Composition of the Rat Sciatic Nerve Institute of H. SCHMALBRUCH Neurophysiology, University of Copenhagen, The Panum Institute, DK 2200 Copenhagen N, Denmark ABSTRACT The rat sciatic nerve originates from the spinal segments L4-L6. It is unifascicular at the trochanter; 5-7 mm distally, the nerve splits into two and then into four fascicles. The tibial portion gives rise to the tibial and the sural nerves, and the peroneal portion gives rise to the peroneal nerve and a cutaneous branch that perforates the lateral hamstring muscles to innervate the proximolateral face of the calf. The number and type of the axons in these branches were determined in light and electron micrographs of normal nerves, and after de-efferentiation or sympathectomy. Deafferentiation was technically not feasible because spinal ganglia and ventral roots were supplied by the same vascular plexus. The tibial nerve contained 1,000 motor and 3,500 myelinated afferent axons, 3,700 sympathetic axons, and 5,400 unmyelinated afferent axons. The peronal nerve contained 600 motor and 1,300 myelinated afferent axons, 1,100 sympathetic axons and 3,000 unmyelinated afferent axons. The sural nerve contained 1,100 myelinated and 2,800 unmyelinated afferent axons; in addition, there were 1,500 unmyelinated sympathetic axons. The cutaneous branch consisted of 400 myelinated and 1,800 unmyelinated afferent axons. Thus, the entire sciatic nerve a t midthigh is composed of about 27,000 axons; 6% are myelinated motor axons, 23% and 48% are myelinated and unmyelinated sensory axons, respectively, and 23% are unmyelinated sympathetic axons. The techniques used did not demonstrate sympathetic axons in the cutaneous branch and did not reveal the few motor axons contained in the sural nerve. The sciatic nerve branches of cat and rat are frequently used in experimental studies. Boyd and Davey (1966, 1968), in a now classical study, counted the myelinated axons in a large number of nerves of cat and, using surgical deafferentiation or de-efferentiation, determined the percentages of motor and sensory fibers. Comparable data for rat nerves do not exist. Experimental studies of rat nerves may contain counts of myelinated axons in the normal sural and tibial nerves (for references, see Peyronnard and Charron, 1982 and Mayhew and Sharma, 1984a), but usually the nerves were taken from the calf region, possibly after branching had commenced. Recently, Jenq and Coggeshall(1984,1985) reported counts of myelinated and unmyelinated fibers in the sural nerve, and in the entire sciatic nerve just after its exit from the pelvis. The results will be discussed below. Separate counts of sympathetic and unmyelinated sensory fibers to my knowledge do not exist. The present paper is a n anatomical study of the fiber composition of the four principal branches that arise from the rat sciatic nerve in the thigh region. The number of motor fibers was determined by de-efferentiation, and the contribution of sympathetic fibers to the pool of unmyelinated fibers was determined by sympathectomy. MATERIALS AND METHODS Forty male Wistar rats were used, each weighing about 300 gm. The fiber counts reported in this paper were (c) 1986 ALAN R. LISS, INC. derived from 18 animals. Nerve fibers of different origin were identified by cle-efferentiation or sympathectomy. For de-efferentiation, 4-week-old rats were used because in younger rats laminectomies are technically easier. All operations were performed in fentanyl-apozepam anesthesia; for the terminal perfusion fixation, the rats were anesthetized with Halotha+. Surgery was performed under clean but not sterile conditions; infections did not occur. De-efferenriation Three to four lumbar laminectomies were performed on the right side including the spinal processes. The spinal ganglia were identified; the crest of the ilium that is closest to L5 served a s landmark. The ventral roots L4-L6 were cut close to the ganglia and bent upward to prevent regeneration. The spinal cord was covered with a hemostatic sponge, and muscles and skin were sutured separately. The rats were sacrificed after 4 weeks. The deficit of myelinated fibers in the sciatic nerve compared to the contralateral side reflected the number of efferent motor fibers. Deafferentiation, i.e., excision of the spinal ganglia L4-L6, always resulted in paresis of leg muscles. The operation caused profuse bleeding from the site where Received August 6, 1985; accepted December 18, 1985. 72 H. SCHMALBRUCH the ventral root came close to the ganglion, and it was difficult to identify both structures. To elucidate the cause of this difficulty, spaced serial sections of the ganglion-ventral root complex were investigated by light microscopy. Syrnpathectorny After laparotomy the abdominal aorta and vena cava were approached from the left side and mobilized, but not separated. Both sympathetic chains were found dorsal to the renal blood vessels and were excised from here to the aortic bifurcation. This corresponds to L2 to S2 (Suh et al., 1984). To ensure completeness, the ventral surfaces of the spinal column and of the prevertebral muscles were cleaned, and all connections to the connective tissue around the large vessels were severed. Care was taken not to injure the left ureter and the dorsal vascular branches supplying the spinal cord. It did not appear to be possible to completely excise one chain alone without injuring the contralateral one. The abdominal wall was sutured in layers. The rats were sacrificed after 10-12 days, which suffices for the unmyelinated axons to degenerate (Coggeshall et al., 1980; Suh et al., 1984). Specimen Preparation The nerves were fixed in situ by perfusion of the hind limbs via the abdominal aorta. The vascular bed was rinsed for 2 min with Ringer’s solution containing heparin (5,000 unitditer) and procaine (0.1 m i t e r ) ; perfusion was continued for 10-12 min with 2.5% glutaraldehyde in Ringer’s solution (1,000 ml, pH 7.4, 20°, 350 mOsM). The nerves remained in the glutaraldehyde solution a t 4°C overnight; the individual branches were postfixed in 1%Os04 in phosphate buffer, and embedded in Embed 812. For light microscopy, cross sections 1- to 3-pm-thick were stained with p-phenylenediamine. For electron microscopy cross sections of a n entire branch were collected on 150-mesh grids without support film, and were stained with methanolic uranyl acetate (Landon, personal communication) and aqueous lead citrate. The samples used to study the topographical relation between spinal ganglia and ventral roots (see above) were taken from normal rats that had been fixed by vascular perfusion through the left cardiac ventricle. Counts and Measurements The myelinated nerve fibers were counted on light micrographs (objective Zeiss Planapo 40 oil; A: 1.0) printed to 1,100 x or 1,800 x . In small nerves all fibers were counted; in nerves with more than 1,000 fibers 3070% of the fibers were counted and the total number was computed from the sample area and the total area of the nerve cross section. The micrographs to be assessed were taken nonoverlappingly according to a scheme that covered the entire cross section. On the same prints, the outer perimeters of the myelin sheaths were measured and divided by 3.14 to obtain “idealized diameters” (see Sources of Error, below). For electron microscopy, one complete cross section of a nerve branch was selected, and a series of micrographs was taken at 3,600 x according to a scheme that covered the entire cross section. The total area photographed corresponded to a t least 3% of the total cross-sectional area of the nerve; in small nerves up to 70% of the area was photographed. Areas with artifacts, contamination, or blood vessels were not excluded. Myelin sheaths and unmyelinated axons were counted on prints at 10,800 x ; structures bisected by two of the four margins were included. The total number of unmyelinated axons was calculated from their relative frequency compared to myelin sheaths, and the total number of myelinated fibers, which already was known from light microscopy (see Sources of Error). The “idealized diameters” of the unmyelinated axons were obtained from their perimeters measured at 26,500 X. The magnifications of light and electron micrograph prints were calibrated against gratings (100 lines per 1 mm, and 2,160 lines per 1mm, respectively), which were photographed and printed together with each series of micrographs. All counts and measurements were done with the aid of a digitizer tablet (Kontron MOP 2) connected to a computer (Digital PDP 11/34). The effect of the hysteresis of the lenses of the electron microscope on its magnification was eliminated by exciting all lenses maximally before focusing; the focus setting was not readjusted during a series of micrographs. Sources of Error “Idealized diameters” of the nerve fibers were calculated from their perimeters because the circumference in contrast to the area is generally independent of the irregular configurations of the cross section (Friede and Samorajski, 1967). Nevertheless, this precaution, in hindsight, turned out to be unnecessary. The mean diameters of myelinated fibers were found to differ by at most 0.1 pm when they were calculated from the perimeter and from the cross-sectional area. This indicates that the effect of distortion was negligible. The micrographs used for the light microscope fiber counts covered a t least 30% of the cross-sectional area of the nerve, and sampling bias was avoided by spreading the micrographs systematically over the entire cross section. Mayhew and Sharma (1984a,b), who tested different sampling methods for the rat tibia1 nerve, found that the gain in accuracy is small when more than 6% of the fibers are assessed, and that the sampling procedure has little effect. Sampling was critical, however, when relatively large nerve branches were studied by electron microscopy. The unmyelinated axons were clustered and a micrograph covering a 350-pm2 cross-sectional area exposed 2-11 (mostly 5-7) myelin sheaths and 0-80 unmyelinated axons. To ensure randomness, the photographs were taken according to a predetermined scheme without inspection of the site to be photographed, and a n equal number of micrographs was placed upon each grid square. The areas hidden by the grid bars were assumed to be randomly selected. Initially the numbers of both unmyelinated and myelinated axons were calculated from counts in electron micrographs, the area assessed, and the total cross-sectional area of the nerve branch measured by light microscopy. The result for myelinated fibers was then compared to counts in light micrographs; this procedure should ascertain that the areas studied by electron microscopy were representative. Unexpectedly, in each nerve the number of myelinated fibers was 10-20% higher when it was determined by electron microscopy than when determined by light microscopy alone. Re- LH!L peated photographing and counting, or counting axons, or counting centers of axons rather than myelin sheaths, gave results identical within f5%. The difference in the number of myelinated fibers could not be attributed to the presence of very thin fibers that might have been overlooked in light micrographs. This indicated that the density of niyelinated fibers (number per unit area) was indeed higher in electron than in light micrographs. Section compression might represent part of the explanation, but it is most likely that the sections were not stretched across the grid holes but that they sagged or scallopped. Folds often disappeared when the sections were exposed to the electron beam, and a t high magnification differences of the section position in relation to the focal plane were seen when different sites were inspected. To account for a 20% error in area, a 150-pm by 150-pm square of the grid would have to expose a 164-pm by 164-pm square of the section; this does not appear unlikely. To circumvent the error in area, the total number of unmyelinated fibers was calculated from their frequency in relation to myelinated fibers, and the counts of myelinated fibers in light micrographs. RESULTS Branches of the Sciatic Nerve The anatomy of the rat has been described by Greene (1963).Nevertheless, the branching pattern of the sciatic nerve of the rats of this study differed in several aspects from that found by Greene (1963). The segmental nerves L4, L5, and L6 contributed to the sciatic nerve; no contribution of L3 and S1 was found. None of the nerves L4-L6 were exclusive to the sciatic nerve. After the sciatic nerve had left the pelvis and had given rise to several branches to the glutei and hamstring muscles, it curved around the greater trochanter. At this site it was always unifascicular. At a point 3-5 mm distal to the greater trochanter a septum dividing the future main branches of the nerve occurred, and a t about the same level a thin branch originated from the peroneal portion of the nerve. This nerve branch entered the lateral hamstring muscle group and without branching, perforated the dense connective tissue between the anterior and posterior heads of the biceps femoris muscle and innervated the skin of the proximolateral face of the calf. In the following this nerve is referred to as “cutaneous branch.” At a point distal to the point where the tibial and peroneal portion of the sciatic nerve separated, the sural nerve originated from the dorsal face of the tibial nerve. Cross sections at the middle of the thigh might show four nerve branches: the large tibial nerve, the smaller peroneal nerve, the still smaller sural nerve, and the very small cutaneous branch (Fig. 1).The peroneal nerve on its way through the thigh gave off two to four very thin nerve branches, each consisting of 10-20 myelinated and several hundred unmyelinated nerve fibers. In respect to fiber composition these nerve branches did not resemble muscular branches and probably innervated the femur and the knee joint. These nerve branches were seen in serial cross sections but could not be identified by dissection. Within the fat tissue filling the popliteal fossa the three remaining branches of the sciatic nerve divided. The sural nerve ran superficially through the middle of the popliteal fossa and, together with prominent blood vessels, continued on the dorsal face of the gastrocnemius KXI b L l A l l L N h K V h /Y muscle; the tibial nerve ran through the depth of the popliteal fossa and disappeared between the heads of the gastrocnemius muscle to innervate the flexor muscles and eventually, by means of the plantar nerves, the palm of the foot; the peroneal nerve took a superficial and lateral course and entered the anterior muscle group. Myelinated Fibers The normal number of myelinated fibers in the sciatic nerve was determined in 18 rats. Four of these were control rats, in nine sympathectomy had been performed, and in five the contralateral sciatic nerve had been de-efferentiated. Sympathectomy or contralateral de-efferentiation did not affect the myelinated fibers, and the results were pooled (Table 1).The entire sciatic nerve contained 7,800 fibers, of which 4,500 formed the tibial nerve and 1,900 the peroneal nerve. In these branches the variation with respect to fiber numbers was small and the interindividual standard deviations were less than 10% of the mean values. The sural nerve and the cutaneous branch contained 1,050 and 350 fibers, respectively. The large interindividual variation of these nerves probably reflected the variability of the sensory innervation of the skin. Four weeks after de-efferentiation, the number of fibers in the right tibial nerve was reduced by about 1,000, and that of the peroneal nerve was reduced by about 600 (Table 2). At that time, almost no degenerating myelin sheaths were left, and no regenerating fibers were found. The loss of myelinated axons after de-efferentiation reflected the numbers of motor fibers in these two nerves. No fiber deficit occurred in the cutaneous branch or in the sural nerve, although the latter nerve in rat contains about 80 motor fibers (Peyronnard and Charron, 1982). The diameters of the myelin sheaths ranged from 1.5 pm to 12.5 pm. The distribution was unimodal in the tibial and peroneal nerves, whereas the sural nerve and the cutaneous branch showed discontinuous distributions. The sural nerve contained a larger proportion of thick fibers than the cutaneous branch (Fig. 2). The shape of the diameter histograms changed considerably during the process of sampling; in places distinct bimodal distributions were found. These areas might have contained groups of fibers determined to form motor branches; the bimodal shapes of the histograms always disappeared with increasing sample size. The diameter distribution of the motor fibers was obtained by constructing “difference histograms.” For each size class the number of fibers in the de-efferentiated nerve was subtracted from the number of fibers in the normal contralateral nerve. In two of three rats the difference histograms of the tibial nerves were bimodal; in one it was skewed (Fig. 3). Unmyelinated Fibers The total number of unmyelinated axons was about 19,000. In all, five nerves were assessed; three were from normal rats and two were control nerves from rats in which the contralateral side had been de-efferentiated. The interindividual variation in all nerve branches was larger than that of myelinated fibers; the standard deviations were about 20% of the mean values. In the tibial and peroneal nerves there were about twice as 74 H. SCHMALBRUCH Fig. 1. The branching pattern of the rat sciatic nerve. A. Lateral view of the right thigh enlarged 1.8 times. The gluteal and lateral hamstring muscles and the fat tissue filling the popliteal fossa have been removed. The position of the trochanter and the knee joint are marked by pushpins labeled t and k, respectively. The cutaneous branch (c) has been cut. Below left the gastrocnemius muscle is seen. 1,2,3) The arrows mark the sites of the cross sections shown in panels B,C,D. B. Cross section of the sciatic nerve at site 1. C. Cross section of the sciatic nerve at site 2. The nerve starts to divide into its tibial and peroneal portions. D. Cross section at site 3. The tibial portion of the nerve has divided into the tibial (t) and the sural (s) nerves, and the peroneal portion has divided into the peroneal nerve (p) and the cutaneous branch (c). FIBER COMPOSITION O F THE RAT SCIATIC NERVE 75 TABLE 1. Number of myelinated axons in sciatic nerve branches of normal rats or in rats after experimental procedures not affecting the myelinated nerve fibers Tibial n. Rat No. Control animals 1 2 3 4 (SD) Cutaneous branch Total 1,754 1,806 1,865 1,611 1,058 979 1,099 884 272 438 328 352 7,309 7,085 8,369 7,114 4,651 4,945 4,647 5,472 4,781 4,266 4,594 4,208 4,985 1,882 2.123 1:964 2,139 1,994 1,592 1,975 1,815 1,973 1,091 1,017 1.417 lj212 833 834 1,049 957 979 466 469 349 427 281 346 397 309 341 8,090 8,554 8,377 9,250 7,889 7,038 8,015 7,289 8.278 4,474 3,999 4,391 4,870 4,124 4,547 (422) 1,797 1,760 1,912 1,952 1,765 1,871 (150) 1,066 1,070 1,123 1,081 1,217 1,054 (142) 348 407 287 375 183 354 (74) 7,685 7,236 7,713 8,278 7,289 7,825 (622) Contralaterally de-efferentiated 14 15 16 17 18 Mean Sural n. 4,225 3,862 5,077 4.267 Sympathectomized 5 6 7 8 9 10 11 12 13 Peroneal n. TABLE 2. Loss of myelinated nerve fibers after de- efferentiation’ Cutaneous Tibial n. Peroneal n. Sural n. Rat 14 1,027 605 149 branch Lost Rat 15 Rat 16 Rat 17 Rat 18 Mean (SD) 948 921 985 1,189 1,014 (106) 549 485 700 601 588 (79) 31 ( - 6912 ( - 136) 387 25 (-52) 17 (-128) - - ‘Most data from Schmalbruch, 1984. 2Note: (-69) means that the de-efferentiated nerve contained 69 more nerve fibers than the contralateral nerve. many unmyelinated as myelinated fibers; in the sural nerve and the cutaneous branch the ratios were 4.1:l and 5.3:1,respectively (Fig. 4). Sympathectomy reduced the number of unmyelinated fibers to roughly 13,000,i.e., the branches of the sciatic nerve together contained 6,000 efferent (sympathetic) and 13,000afferent (sensory) unmyelinated axons. The absolute loss of unmyelinatd fibers was largest in the tibia1 nerve; 3,700,or 40%, of the unmyelinated axons were sympathetic. The deficit in the peroneal nerve was 1,100,which corresponded to 27% of all unmyelinated axons; in the sural nerve it was 1,500,corresponding to 35% of the unmyelinated axons (Table 3). Sympathectomy did not produce a significant loss of unmyelinated fibers in the cutaneous branch. The diameter distribution of the unmyelinated axons was unimodal in all nerves. The peak of the histograms was at 0.7-0.8pm; the standard deviation of the axon diameters was 20-25% of the mean. After sympathectomy, the mean diameters tended to be reduced. “Difference histograms” (see above) revealed that not only large but also small axons had been lost. There was no change in diameter in the cutaneous branch after sympathectomy, suggesting that this nerve contained no or only few autonomic fibers 4). Topography of the Ventral Root-Spinal Ganglion Complex Cross sections of plastic-embedded samples for light microscopy showed that the ventral roots were practically devoid of a connective tissue sheath. The ventral root L5,which was the thickest one of the three roots to the sciatic nerve (Schmalbruch, 1984),had a diameter of about 400 pm. When it approached the ganglion both became enclosed by a common connective tissue sheath about 10 pm thick. Cross sections of samples fixed by vascular perfusion demonstrated a vascular plexus that supplied the ganglion and the ventral root; there were also vessels in the 20-pm-wide gap between the two structures. Before the distal pole of the ganglion, peripheral afferent and efferent ventral root fibers became so close that even in cross sections it was impossible to delineate afferent and efferent structures without the aid of proximal serial sections (Fig. 5). The situation at the ganglia L4 and L6 was the same as at L5; the ventral root L6 was much thinner than the L4 or L5 roots. DISCUSSION The rat sciatic nerve in the thigh, distal of the greater trochanter, consists of 7,800 myelinated and 19,400unmyelinated axons. Of these, 1,600myelinated and 6,400 unmyelinated axons are efferent, and are motor axons and sympathetic axons, respectively (Tables 1-3,5). The ratio unmyelinated afferent axons to myelinated afferent axons is 2 2 1 , and, hence, smaller than the corresponding 3:l ratio in thoracic dorsal roots (Hulsebosch and Coggeshall, 1983). Whether this reflects different cell populations in lumbar as compared to thoracic dor- H. SCHMALBRUCH 76 rat 2 rat 1 tibial n n 1219(=32%) tibial n. n 688(=16%) 10 h peroneal n. n 548(=30%) sura n . n 58 (=55%) sural n. n 452(=46%) iI 15 0 peroneal n . cut. branch 1 100%) cut. branch n 438(=100%) unmyelinated fibers. Their data for myelinated fibers are in harmony with those reported here (Table 1).It is interesting that the number of myelinated fibers does not increase when the nerve splits up into its branches; i.e., that there is no branching within a distance of at least 10 mm. Their counts of unmyelinated fibers in both nerves, however, are almost 20% less than in the present study (Table 3). This might suggest axonal branching in the sciatic nerve. Nevertheless, this assumption does not explain the difference in the sural nerve; in addition, our own unpublished experiments indicated that, within a 20-mm-long segment of the peroneal nerve, the unmyelinated axons do not branch. Jenq and Coggeshall(1984, 1985)counted nerve fibers in photomontages of electronmicrographs of the entire nerve. The final magnification is not given, but it seems reasonable to assume that it was less than 10,000 x . In my experience, it is difficult to identify the smallest axons below a magnification of 10,000. The sciatic nerve gives rise to four nerve branches (Fig. 1;Table 5). Its two largest branches, the tibial and peroneal nerves, supply the dorsal and ventral compartments of the lower leg, and contain 1,000 and 600 motor axons, respectively. The motor percentage of these nerves (22% and 31%) is much smaller than that reported for cat nerves by Boyd and Davey (1968) (4070%). Nevertheless, tibial and peroneal nerves at their origin from the sciatic nerve still contain many skin afferents, whereas Boyd and Davey (1968)preferentially studied muscular branches. It is unlikely that the number of motor axons in the present study was underestimated. By using horseradish peroxidase (HRP)as a label, Brushart and Mesulam (1980)found 866 tibial motoneurons (one rat) and 368-434 peroneal motoneurons (six rats). The 80 motor axons of the sural nerve, shown by Peyronnard and Charron (1982)by HRP labeling and by deafferentiation (see below), did not cause a consistent fiber loss after de-efferentiation in this study (Table 2). Nevertheless, it is unquestioned that the sural nerve of rat contains motor axons tht eventually enter the lateral plantar nerve (see, for example, Betz et al., 1979). The “cutaneous branch” apparently is purely afferent, but the large interindividual variation of the size of this nerve might have obscured the loss of efferent unmyelinated fibers after sympathectomy. It is well established that the ventral roots of mammals may contain myelinated and unmyelinated sensory fibers (for references, see Coggeshall et al., 1974). These sensory fibers were severed during de-efferentiation as well. Nevertheless, this did not influence the fiber counts in the peripheral nerves. Sectioning of the sensory ventral root fibers causes degeneration of the central segments only, whereas the distal segments and hence also the ganglion cells survive (Coggeshall et al., 1974). The diameter of myelinated axons in rat is smaller than in cat. Boyd and Davey (1968) measured fiber diameters of up to 18 pm, whereas in rat only a few fibers are 12 pm thick. This species difference seems real, despite differences in the histological technique: Boyd and Davey (1968) studied paraffin sections 7 pm thick, whereas in this study plastic sections 1-3 pm thick were used. The histograms of fiber diameters in all intact and de-efferentiated tibial and peroneal nerves are unimodal ’L I‘LL 7 0 2 4 6 8 1012 tJm 0 2 4 6 8 1 0 1 2 Prn Fig. 2. Diameter distribution of the myelinated fibers in the four sciatic nerve branches of rats 1 and 2. n, number of axons measured (= 410 of all axons of the nerve branch). The diameters range from 2 pm to 12 pm. The distributions are unimodal with peaks at about 8 pm in the tibial and peroneal nerves, but appear discontinuous in the sural nerve and the cutaneous branch (see also Table 1). sal roots, or is due to branching of myelinated afferents, is unknown. Jenq and Coggeshall (1984, 1985) found in the rat sural nerve 1,062 myelinated and 3,557 unmyelinated fibers; the unifascicular most proximal segment of the sciatic nerve contained 8,120 myelinated and 15,542 R A T 16 R A T 17 C O N T R O L u %IBi m 20 R A T 18 1 12 n= 1 7 6 5 24 i A ' F F E R E N k?!+ n= 1 2 5 2 200 r " E1F F E R E N 1T " n 150 100 50 0 u - 0 2 l 4 l 6 l 8 1 P l 0 l 0 I I 1 l 2 4 Fig. 3. Diameter distribution of the myelinated fibers in the normal (top) and the de-efferentiated (middle) tibia1 nerves of rats 16, 17, and 18. The bottom-row histograms were constructed by subtracting the upper two histobvams from each other to obtain the diameter distri- 6 J 8 1 0 0 P I 2 1 4 1 6 I 8 I 1 0 P bution of the eliminated efferent fibers. Note that the class frequency in the two upper rows is given in percentage of all axons; the scale for the bottom row indicates the computed number of axons in each class. n, total number of myelinated axons. (See also Tables 1 and 2). TABLE 3. Number of unmyelinated axons in sciatic nerve branches of normal rats and after sympathectomy Rat No. Normal 1 2 3 14 15 Mean (SD) Sympathectomized 5 7 9 10 11 12 13 Mean (SD) Peroneal n. Sural n. Cutaneous branch 8,640 6,742 9,918 11,049 9,037 9,077 (1,600) 4,155 3,351 4,795 3,967 4,588 4,171 (565) 4,209 3,773 3,654 lost 5,590 4,307 (888) 1,752 2,198 2,100 2,176 1,176 1,880 (433) 18,756 16,064 20,467 20,391 18,9201 (2,061) 7,821 5,913 3,075 4,173 5,642 7,579 3,624 5,404 (1.871) 2,963 2,771 3,631 2,548 2,583 3,938 2,885 3,046 (544) 2,073 3,812 2,911 2,594 2,811 2,668 2,660 2,790 (523) 2,523 1,344 1,625 2,541 2,070 1,205 1,357 1,809 (568) 15,380 13,840 11,242 11,856 13,106 15,390 10,526 13,048 (1.940) Tibia1 n. Total 'The mean total number of unmyelinated axons calculated from the mean numbers of axons in each nerve branch is 19,435;this explains the discrepancy between this figure and the data given in the text. 78 H. SCHMALBRUCH Fig. 4. Typical electron micrographs of cross sections of the normal peroneal nerve (A) and the cutaneous branch (B).The peroneal and also the tibia1 nerves consist of rather large axons with interspersed groups of unmyelinated axons. In the cutaneous branch and also in the sural nerve large areas of the cross section are occupied by unmyelinated axons. (Fig. 2). In the rat L4 dorsal root the fiber distribution is unimodal; it is bimodal in the L4 ventral root (Rao and Krinke, 1983). The shape of the histogram in the intact nerve is probably dominated by the large number of afferent fibers, whereas the “difference histograms,” reflecting the diameter distribution of the efferent fibers, tend to be bimodal (Fig. 3). Muscular branches in rat always show a bimodal diameter distribution (Zelena and Hnik, 1963; Eisen et al., 1974).The diameter distribution of the unmyelinated nerve fibers in all nerves is unimodal and remains so after sympathectomy; preferentially but not exclusively, large fibers are lost. This Fig. 5. The anatomical relation between the L5 ventral root and the L5 dorsal root ganglion shown in stepped serial cross sections. A. The ventral root proximal to the ganglion. The circular defects represent blood vessels ballooned by perfusion fixation. B. The ventral root and the ganglion are enclosed by a common connective tissue sheath about 10 pm thick. C . The gap between ganglion and ventral root is about 20 pm wide and contains blood vessels. Note the large number of afferent fibers within the ganglion, close to the ventral root. D. Towards the distal pole of the ganglion fascicles of peripheral afferent axons and efferent ventral root fibers are difficult to delineate without the aid of proximal sections. There were numerous ganglion cells outside the area depicted. There seems to exist a discrepancy between the anatomical situation in the rat shown in these micrographs, and the reports of complete surgical deafferentiation, assuming that this operation inflicted no or only little damage on the ventral roots (see text). H. SCHMALBRUCH 80 TABLE 4. Number and diameter of the unmyelinated axons in branches of the sciatic nerve of a normal rat as compared to a rat in which the autonomic fibers have been eliminated’ P Sural n. Mean diam. (pm) Cutaneous br. Mean diam. SD N (pm) (pm) (pm) N 8,640 0.80 0.19 4,155 0.78 0.18 4,209 0.77 0.16 1,752 0.73 0.14 5,642 0.75 0.20 2,583 0.74 0.18 2,811 0.70 0.16 2,070 0.72 0.14 N Rat 1, normal Rat 11, sympathectomized Peroneal n. Mean SD diam. (pm) (pm) Tibial n. Mean diam. (pm) SD < 0.1 N < 0.05 SD (pm) < 0.5 <0.001 ‘Note that there is no change in the cutaneous branch. SD, standard deviation; P, significance limits of the diameter difference according to Student’s t test. TABLE 5. Fiber composition of the rat sciatic nerve branches: Summary of results presented in Tables 1-3’ Tibial n. Efferent Myelinated motor axons Unmyelinated sympathetic axons Afferent Myelinated axons Unmyelinated axons Peroneal n. Sural n. Cutaneous branch - - 1,000 600 3,700 1,100 1,500 (ioo? 3,500 1,300 1,100 400 5,400 3,000 2,800 1,800 ‘Data are mean values to nearest hundred. 2Not significant. suggests that the sympathetic fibers on the average are thicker than unmyelinated afferent fibers (Table 4). It would have been desirable to deafferentiate the nerves by excision of the appropriate ganglia in order to assess the number and size of the motor axons in a direct approach. This turned out to be impossible (Fig. 5). Muscle paresis regularly occurred after attempts to excise the ganglia; it was probably due to direct trauma during the operation and also to ischemia following the destruction of the vascular plexus supplying ventral root and ganglion. Connective tissue that would allow separation of efferent and afferent structures in a blunt fashion is scarce. Wray (1969), who determined the number of motor units in the much larger baboon, encountered similar difficulties and often was forced to use de-efferentiation rather than deafferentiation. Nevertheless, Zelena and Hnik (1963), Gutmann and Hanzlikova (19661, and Peyronnard and Charron (1982, 1983) determined in rats the number of motor axons in deafferentiated nerves of the soleus and anterior tibial muscles, and in the deafferentiated sural nerve. Zelena and Hnik (1963) and Peyronnard and Charron (1982, 1983) do not report technical difficulties. Gutmann and Hanzlikova (1966) cut only the dorsal roots, but did not discuss whether this caused the desired degeneration of afferent fibers in the peripheral nerve. The branching pattern of the sciatic neve in several aspects differed from that found by Greene (19631, although she used Wistar rats as well. According to Greene (19631, the tibial and peroneal portions of the nerve are separatable up to their origin from the plexus; in the rats used for the present study, and also in those investigated by Jenq and Coggeshall(1985), the sciatic nerve was unifascicular at the trochanter. Greene (1963) reports that the sural nerve originates from the peroneal nerve; in the present rats it unequivocally and constantly originated from the tibial nerve. 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