THE JOURNAL OF COMPARATIVE NEUROLOGY 293377-398 (1990)
Anatomical Studies of Dorsal Column Axons and Dorsal Root Ganglion Cells After Spinal Cord Injury in the Newborn Rat STEPHEN P. LAHR AND DENNIS J. STELZNER Department of Anatomy and Cell Biology, SUNY Health Science Center, Syracuse, New York 13210
ABSTRACT The response of dorsal column axons was studied after neonatal spinal overhemisection injury (right hemicord and left doral funiculus). Rat pups (N = 11) received this spinal lesion a t the C2 level within 30 hours after birth. The cauda equina was exposed 3 months later in one group of chronic operates ( N - 5) and in a group of normal adults ( N - 2), and all spinal roots from IA caudally were cut bilaterally; 4 days later the spinal cord and medulla were processed for Fink-Heimer impregnation of degenerating axons and terminals. In a second group of chronic operates ( N = 6) and normal adult controls (N = 4)the left sciatic nerve was injected with a cholera toxin-HRP conjugate (C-HRP), followed by a 2-3 day transganglionic transport period, and then the spinal cord and medulla were processed with tetramethylbenzidine histochemistry. Both control groups have B consistent dense projection in topographically adjacent regions of the dorsal funiculus and gracile nucleus. However, there is no sign of axonal growth around the lesion in either group of chronic experimental operates. Instead, there is a decreased density of projection within the dorsal funiculus near the lesion site. Many remaining C-HHP labeled axons in the experimental operates have abnormal, thick varicosities and swollen axonal endings (5-10 pm x 10-30 pm) within the dorsal funiculus through several spinal segments caudal to the lesion. Ultrastructural analysis of the dorsal funiculus in three other chronic experimental operates reveals the presence of numerous vesicle filled axonal profiles and reactive endings which appear similar to the C-HKP labeled structures. Transganglionic labeling after C-HRP sciatic nerve injections (N = 4) and retrograde labeling of L4, L5 dorsal root ganglion neurons after fast blue injections of the gracile nucleus ( N = 6) both suggest that all dorsal column axons project to the gracile nucleus in the newborn rat. Dorsal root ganglion (DRG) cell survival following the neonatal overhemisection injury was also examined in the L4 and L5 DRG. DRG neurons that project to the gracile nucleus were prelabeled by injecting fast blue into this nucleus a t birth two days prior to the cervical overhemisection spinal injury. Both normal littermates (N= 9) and spinally injured animals (N = 12) were examined after postinjection survival periods of 10 or 22 days. Comparison of the percentage
Accepted September 28,1989. S.P. Lahr’s present address is Department of Anatomy, Medical College of Pennsylvania, 3200 Henry Ave., Philadelphia, PA 19129. Acknowledgments: We would like to thank Ms. Judith Straws for technical advice and photographic assistance, Ms. Maria Shurant for electron microscopic sectioning and preparation, nr. Carolyn Rates for her critical reading of the manuscript, and Ms. Nancy Snyder for typing. Supported by grant NS14096.
0 1990 WILEY-LISS, INC.
S.P. LAHR AND D.J. STELZNER
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and number of labeled and unlabeled DRG neurons in the L4 and L5 ganglia in control and experimental operates reveals no sign of cell loss after neonatal dorsal column axotomy. We conclude that dorsal column axons do not grow around damage to their normal pathway in the newborn rat during a period when previous studies show adjacent corticospinal axons are able to grow around the same type of spinal injury and when the spinal environment normally permits growth of the still elongating corticospinal tract axons to occur. Key words: CNS regeneration, CNS development, retrograde cell death, transganglionic transport, fluorescent neuronal labeling
The neonatal rat spinal cord has several features that make it very useful as a model for studying mammalian central nervous system (CNS) regeneration. Many factors thought to interfere with axonal regeneration in the adult CNS are minimal or absent. The neonatal rat spinal cord undergoes minimal glial scarring after injury (Prendergast and Stelzner, '76a; Barrett et al., '84), all pathways are unmyelinated (Matthews and Duncan, '71), and most of the glial population is horn postnatally (Gilmore, '71). Phagocytosis after injury is very rapid, being complete by the third postoperative ( p a ) day (Gilbert and Stelzner, '79). Moreover, factors involved in forming specific nerve connections still persist during this period, since axonal elongation and synaptogenesis occur postnatally for certain pathways. One further advantage to study of the developing spinal cord is that various ascending and descending pathways are at different states of maturity a t birth. Therefore, more than one pathway can be cut a t the same time and the ability of each to elongate can be compared when local factors a t the lesion site should he similar. The (CST) is a late developing pathway in the rat (Donatelle, '77; Schreyer and Jones, '82). The majority of growing CST fibers are localized in the ventral portion of the dorsal funiculus contralateral to their origin just as in the adult. The CST fibers do not reach the upper cervical spinal cord until birth, grow into the thoracic segments by day 3, and reach the lumbar cord by the end of the first postnatal week. Growth of the CST into the dorsal horn occurs 2 to 3 days after the main pathway reaches a given segmental level. A mature distribution of CST axons and the development of hindlimb tactile placing indicates that synaptogenesis is completed at the beginning of the third postnatal week (Donatelle, '77). When the neonatal rat spinal cord is overhemisected (cutting the dorsal funiculus bilaterally), at cervical or thoracic levels, previous work from this laboratory (Bernstein and St,elzner, '83; Bates and Stelzner, '87; Bates et al., '88) and work by Schreyer and Jones ('83) shows fibers from the postnatally elongating CST will grow around the lesion, extend caudally in an aberrant pathway, and terminate in normal projection zones through postnatal day 6. A high cervical lesion made on postnatal day 3 or later is presumably after the period when most growing CST axons have passed through the level of injury, although the possibility of a population of late growing axons has not been ruled out. Bregman and Goldberger ('82, '83) have shown that the CST will grow around a spinal hemisection made in young kittens. The work of Tolbert and Der ('87) in the kitten suggests that
growth around early spinal injury in this species is only by late growing axons. In the pouch young opossum, damage to the region of the spinal cord through which the rubrospinal tract will normally grow also results in rubrospinal axons growing around this lesion, but this growth is not seen in the more mature animal after rubrospinal growth is completed (Xu and Martin, '89). Kalil and Reh have cut one pyramidal tract 2-3 mm rostra1 to the pyramidal decussation in hamsters during the first postnatal week, and have shown that CST fibers will grow around the lesion (Kalil and Reh, '79, '82). These fibers descend in an aberrant but consistent pathway, and terminate in the normal CST projection area within the cervical spinal cord. The terminations formed by both the normal and aberrant pyramidal tract are similar in their organization within the cervical spinal cord. This anomalous growth is seen most abundantly in hamsters lesioned during the first postnatal week. During normal development the CST projection of the hamster has extended to mid-medullary levels by postnatal day 2, and the full complement of axons is in the pyramidal decussation by postnatal day 3 (Reh and Kalil, '81, '82). This suggests that the aberrant pathway seen following pyramidal injury after the second postnatal day in the hamster is made of regrowing axons, although a proportion of CST neurons undergo post-axotomy cell death after early pyramidal injury (Merline and Kalil, '88). Whether by uncut late growing axons or by regenerating axons, the above-mentioned studies indicate that developing CST axons, still elongating to their target, are able to grow around a partial spinal injury during the early postnatal period in several different species. The dorsal column (DC) projection is adjacent to the CST in the rat. These collaterals of centrally projecting dorsal root ganglion (DRG) axons terminate in the gracile and cuneate nuclei. The dorsal root projection is a much more mature pathway a t birth than the CST. The adult distribution of segmental dorsal root axons and mature synaptic connections are found in the lumbosacral enlargement a t birth (Gilbert and Stelzner, '79). Although it is unknown if all DC axons have arrived in the gracile nucleus a t birth, a projection is apparent from lumbosacral DRG cells prenatally and some synaptic contacts have formed in neonatal rats (Chimelli and Scaravelli, '87). Axons in the gracile fasciculus begin myelination by postnatal day 5 , whereas myelination does not begin in the CST until the end of the second postnatal week (Matthews and Duncan, '71). This juxtaposition of the immature CST and the more mature DC pathway within the dorsal funiculus provides an excellent opportunity to determine the ability of DC axons t o
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DORSAL COLUMN AXOTOMY IN NEWBORN RAT SPINAL CORD
A
Chronic Operate
B
Chronic Overate
Control Adult
Control Adult
D
PD 10 or 22
PD 10 or 22 Overhemisection
Newborn Operate
L4 & 5 DRG Neurons
Count L1& i. DUG Neurons
Fig. 1. Diagrammatic illustration of experiments 1.A.1 (A),1.A.2 (B), 2.B (C), and 3 (D). See Methods for details. PD, postnatal day; DRG, dorsal root ganglion.
grow after partial spinal injury. Dorsal column axons should have a similar opportunity to elongate through the same spinal territorv used by CST axons to grow around this injury. Moreover, the peripheral process of DRG neurons is known to regenerate very well after peripheral nerve injury and the central process of the dorsal root is known to regen-
erate to the central and peripheral nervous system interface after injury in adult animals and to cross into the spinal cord at birth (Ramon y Cajal, '28; Stensaas et al., '79; Carlsted, '85). Thus, the potential ability of DC axons in the newborn rat to regenerate should be relatively high. In the present investigation the response of lumbosacral DC axons and
380 DRG cells to newborn spinal injury, and the maturity of the DC projection at birth was examined to compare with previous data on the rat CST.
METHODS Experiment 1: Tests of dorsal column growth after spinal injury in the newborn rat l.A: Newborn spinal surgerg. Newborn SpragueDawley rat pups (n = 11)received a right-sided spinal overhemisection injury at the C 2 level within 30 hours after birth. Near-term pregnant females were purchased from a local supplier (Taconic Farms), or litters were obtained from breeding colonies maintained in our animal care facility. Neonatal rats were anesthetized with cold. After exposing the first and second cervical vertebrae, an incision was made through the ligaments between the first and second cervical vertebrae to expose the dorsal spinal cord. The dura was cut, transversely, and the dorsal funiculus identified visually with the aid of a Zeiss operating microscope. A small iridectomy scissors was centered and lowered approximately halfway into the cord, cutting through the dorsal ftmiculus completely. The scissors were withdrawn and reinserted to sever the right half of the cord. To ensure that the cord was completely overhemisected, a fine dural knife was drawn across the lesion. The wound was packed with saline soaked gelfoam and the superficial muscles and skin sutured separately with 7-0 silk thread. Upon completion of the operation the pups were placed under a heat lamp until they recovered spontaneous movement and then were returned to the mother. The extent of the lesion was verified in all operates by preparing frozen sections through the lesion area a t 100 pm intervals, counterstaining with cresyl violet, and examining Ihe lesion area at 160x to 400x magnification. Camera lucida drawings were made a t low-power magnification to reconstruct the lesion site serially. l.A.l: Degeneration analysis of dorsal column growth (Fig. I A ) . A group of chronic operates (3 months p.o., n = 5) and a group of normal adult control animals (n = 2) had the cauda equina exposed and all the spinal roots caudal to L4 severed bilaterally after anesthesia with chloral hydrate (350 mg/kg, i.p.). Four days later animals were deeply anesthetized with chloral hydrate and sacrificed by intracardiac vascular perfusion first with 50-100 ml of 0.9 % saline, followed by 300-500 ml of 10% neutral buffered formalin. One to 24 hours later the spinal cord and medulla were dissected out of the vertebral canal and cranium and placed in the perfusion fixative overnight. The medulla and representative spinal segments from the different, regions of the spinal cord were placed in a mold and positioned for consistent orientation, and then embedded in egg yolk which was hardened into blocks by exposure to formaldehyde vapors. Hardened blocks were placed into a solution of 25';; sucrose and 10%;buffered formalin and refrigerated for at least 3 days. Frozen sections of the medulla were cut a t 25-30 wm in the transverse plane and sections from the spinal cord were cut either t,ransversely or longitudinally in the horizontal plane, and then processed with the Fink-Heimer silver impregnation method (Fink and Heimer, '67). Ten to 20 well-stained longitudinal sections, taken at 100 pm int.ervals including the cervical spinal dorsal funiculus, and 10 to 20 transverse sections from segments of the cervical, thoracic, and lumbar spinal cord were examined with
S.P.LAHR AND D.J. STELZNER brightfield microscopy for signs of degeneration argyrophylia from anterogradely degenerating axons and terminals arising from the severed dorsal roots. In addition, approximately 20 transverse sections rostra1 to the spinal lesion were taken at 100-400 pm intervals through the caudal medulla, including the gracile nucleus, and examined for evidence of degeneration argyrophilia. l.A.2: Analysis of dorsal column growth by transganglionic labeling (Fig.1B). A second method to assess DC growth used a sensitive H R P conjugate (Trojanowski et al., '81: Wan et al., '82; Trojanowski, '83) and transganglionic transport (Oppenheim and Heaton, '75; Grant et al., '79) t,o complement the results of the Fink-Heimer method. For this procedure, chronic experimental rats that had received the cervical spinal overhemisection injury three to six months previously (n ti) (as described above), and normal adult controls (n = 4) received an injection of cholera toxin conjugated to H R P (C-HRP; List Biological Laboratories Inc.) into the left sciatic nerve (0.75%; 3-5 pl) (Polistina et al., '87) using a modification of the method used by Jankowska for retrograde transsynaptic labeling (Harrison et al., '84; Jankowska, '85). The left sciatic nerve was selected for labeling because it was considered more likely to have cells with fibers able to grow around the spinal lesion since intact tissue remained on this side of the cord (as in Bernstein and Stelzner: '83). The chronic spinal overhemisected operates and adult control animals were anesthetized with chloral hydrate (350 mg/kg, i.p.) and a curvilinear incision made through the skin over the left hip and posterior thigh to expose the sciatic nerve from mid-thigh up to its exit from the sciatic notch. Approximately 1 cm distal to the sciatic notch a loop of 7-0 silk thread was t,ied tightly around the nerve to completely constrict it, the nerve was cut just distal to the ligature and separated away from the muscle and fascia up to the sciatic notch. One-halfcm proximal to the ligature the nerve was crushed with jewelers forceps using moderate pressure for five to ten seconds. A Hamilton syringe with either a 32 gauge needle or a glass micropipette tip was mounted in a micro-manipulator (Fine Science Tools) and positioned alongside the nerve. The nerve was gently lifted and the portion just. proximal to the ligature was inserted and drawn over the end of the needle or micropipette so that the tip was 2-3 mm within the nerve from the point of entry near the ligature. Three to 5 pI of C-HRP was then injected over a 5 minute period into the nerve within the area between the crush and the ligature. The C-HRP had been previously dissolved into a dilute solution of fast green dye, which enhanced visualization of the nerve filling process. The syringe was left within the nerve for 5 minutes before being removed, and the skin was closed with wound clips. Preliminary studies indicated that a 2 to 3 day period was adequate for transganglionic transport. Following the transport period the animals were again deeply anesthetized and sacrificed by intracardiac vascular perfusion with 50-100 ml of a solution of 0.9:; saline in 0.1 M phosphate buffer (PB) (pH 7.4) followed by 200-400 ml of a solution of 1.0''¶formaldehyde and 1.25% glutaraldehyde in 0.1 M PB saline. One to 2 hours later the spinal cord and medulla were dissected out and placed in a solution of 20", sucrose in PB saline and refrigerated overnight. The medulla and representative spinal segments from cervical, thoracic, and lumbar regions of t,he spinal cord were placed in a mold and positioned for consistent orientation, and then embedded in glutaraldehyde-hardened egg yolk. The blocks of tissue were refrigerated overnight before the me-
DORSAL COLUMN AXOTOMY IN NEWBORN RAT SPINAL CORD dulla and lesion zone of the cervical spinal cord were cut in the transverse plane at 25 pm. Selected segments of the spinal cord were cut either transversely or parasagittaly a t 25 pm and collected in phosphate buffer. Processing of sections from the spinal cord and medulla was carried out with conventional tetramet,hylbenzidine (TMB) histochemistry (Mesulam, '78; Mesulam et al., '80). A series of approximately 20 parasagittal sections, taken at 50 pm intervals, including the cervical spinal dorsal funiculus, were analyzed with both bright- and darkfield microscopy for the presence of axons and terminals labeled with C-HRP reaction product. Transverse sections through the lumbar, thoracic, and cervical spinal segments, the lesion area, and approximately 20 to 30 transverse sections through the medulla, including the gracile nucleus, were also examined. Alternate sections were analyzed aft.er being lightly counterstained with neutral red. 1.A.3: Ultrastructural analysis of surviving dorsal colu m n axons. Chronic operates (n = 3) and normal adults (n = 2) were deeply anesthetized with chloral hydrate and sacrificed b57 intracardiac vascular perfusion with 300 ml of a solut.ion of 2.0'7 paraformaldehyde and 1.070 glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.4 to which 0.2% calcium chloride was added (Weber and Stelzner, '80). One t o 24 hours later the spinal cord was dissected out in toto and placed in the perfusion fixative overnight. Pieces of spinal cord were cut from cervical (including the lesion area in chronic operates), thoracic, and lumbar segments. The segments were trimmed with the aid of an operating microscope, and the dorsal funiculus was isolated to make a 1.5 mm long by 0.5 mm thick block of longitudinally oriented white matter. This block of dorsal funiculus was rinsed in PB for 30-60 minutes, postfixed in osmium for 1--2 hours, and dehydrat.ed in graded alcohols and propylene oxide. The tissue was infiltrated with Epon (Ted Pella, Inc.) overnight at room temperature, changed to fresh Epon, and polymerized at 60°C for 24-48 hours. The Epon blocks from the dorsal funiculus were semithin sectioned parasagittaly at 1 pm thickness and examined with phase microscopy. Selected blocks containing swollen axons or endings were thin sectioned (60-90 nm), mounted on copper grids, counterstained with lead citrate, and examined with a JEOL 100 CX-TI electron microscope for the ultrastructural appearance of these axons. A total of 9 to 12 different cervical and lumbar areas were examined from each chronic experimental or control operate and thin sections cut, using glass or diamond knives.
Experiment 2: Tests of dorsal column maturity in the neonatal rat 2.A: Transganglionic labeling. Newborn rat pups, approximately 18 hours old (n = 4), were anesthetized with cold and injected with C-HRP (0.75%; 0.1-0.2 pl) into the left sciatic nerve as described above in experiment 1.A.2. The animals were allowed to survive 20 to 24 hours for transganglionic transport of C-HRP. After deep anesthetization with ether, they were sacrificed by intracardiac vascular perfusion with 10-20 ml of 0.1 M PR saline (pH 7.4) followed by 50 ml of a solution containing 1.0% paraformaldehyde and 1.25',. glutaraldehyde in 0.1 M PB saline (pH 7.4). The medulla and spinal cord were dissected in toto and embedded in egg yolk blocks as described previously in experiment 1. A 2 Frozen sections of the medulla and representative spinal cord segments from the lumbar and tho-
381
racic cord regions were cut transversely a t 25 pm, collected into PR, and processed with TMB histochemistry. Approximately 15 to 20 transverse sections, taken through the medulla a t 50-100 pm intervals and a t various spinal levels, were analyzed with both bright- and darkfield microscopy for the presence of axons and terminal endings labeled with C-HRP reaction product. These sections were lightly counterstained with neutral red, and particular attention was paid to the medulla cytoachitecture in order to determine the location of the laheled axons. 2.B: Retrograde labeling of lumbar DRG neurons (Fig. IC). The retrogradely transported fluorescent, neuronal cell label fast blue (Bentivoglio et al., '80; Kuypers and Huisman, '84) was injected into the gracile nucleus of newborn (less than 36 hours old) rats ( n 6) and young adult (2-3 months old) rats (n 6). Pilot studies showed that fast. blue was superior to diamidino yellow and fluoro-gold in retrogradely labeling DRG neurons in this experimental paradigm. The rat pups were anesthetized with cold and the adults with chloral hydrate (350 mg/kg, ip.). The rostra1 cervical spinal cord and caudal medulla were exposed and the dura lifted gently and cut to visualize the dorsal columns leading into the gracile nucleus. A Hamilton 1or 5 pl syringe with an attached glass micro-pipette tip was filled with fast blue (3.07;) and attached to a micro-manipulator (Fine Science Tools Inc.). The tip was lowered at a slight mediolateral angle ahout 0.6 mm into the lateral portion of the right gracile nucleus and a small amount of fast blue (0.1-0.2 pl, newborns; 0.5-1.0 pl, adults) was injected slowly into the nucleus. The tip was left in place for five minutes after the injection was completed. The newborn animals were given a 2 day and the adults a 3 day pohnjection survival period for retrograde transport, periods giving adequate time for retrograde transport in pilot studies. The injected animals were then sacrificed as described previously in experiment 1.A.1, except the perfusion fixative for this procedure was a solution of 1091 formalin in phosphate buffered saline. One to 24 hours later the medulla and the right L4 and L5 dorsal root ganglia were dissected out and refrigerated overnight in a solution of l o r r formalin and 20";. sucrose in PB. These ganglia were selected for analysis because the DC projection from the sciatic nerve originates from L4-6 dorsal root ganglion cells in the rat, (Hebel and Stromberg, '86), mostly from L4 and L5. The medulla of each animal was embedded in glutaraldehyde-hardened egg yolk as described previously in experiment l.A.2. The DRG were placed in a mold on a 1-2 mm thick layer of semidried egg yolk and then embedded completely with glutaraldehyde-hardened egg yolk and refrigerated overnight. Frozen sections were cut transversely through the caudal medulla and gracile nucleus at 25 pm, and the DRGs were cut in the longitudinal plane at a thickness of 10 pm. The sections were monnted out of distilled water onto subbed slides and kept covered prior to analy>
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s1s.
The percentage of fluorescently labeled cells in the 124 and L5 DRG was determined as described below (see DRG Cell Analysis) and the injection site in the medulla that was heavily labeled was also det.ermined.
Experiment 3: Test of DRG cell survival after neonatal spinal lesion (Fig. ID) To determine whether DRG cells which project to the gracile nucleus a t birth survive a cervical or thoracic spinal
382 overhemisection injury, these neurons were prelabeled by injection of the gracile nucleus of newborn rats with fast blue as described previously in experiment 2.B. Two days after the fast blue injection, a group of these rats received a spinal cord overhemisection injury (at either cervical or rnidthoracic levels) as described in experiment 1.A and in Bernstein and Stelzner ('83), and the animals were allowed to survive for postinjection periods of 10 (n - 6) and 22 (n = 6) days. The medulla, lesion site, and right L4 and L5 DRG were removed and embedded as described previously in experiment 2.B. The percentage of fluorescently labeled cells in the L4 and L A DRG was determined as described below (see DRC Cell Analysis) and compared to control animals not receiving the spinal injury a t the same postinjection periods of 10 (n = 5) and 22 days (n = 4).
DRG Cell analysis of experiments 2.B and 2.3 T o determine the percentage of cells retrogradely labeled with fast blue, 10 pm frozen sections were dehydrated in graded alcohols, cleared in xylene, and coverslipped with D.P.X. mountant (BDH Limited) and analyzed by light microscopy. .4n average of eight ( *3 S.D.) sections (range of 3-12 sections) were taken a t uniform intervals from the right L4 and L5 DRG. Sections were examined with a Nikon Microphot-FX microscope under ultraviolet epi-illuminatioii. An attempt was made to sample the entire extent of each DRG; but section fragility, etc., sometimes obviated this possibility. In such cases every third to fifth section through regions of the DRG with adequate tissue preservation were analyzed. Each section was examined at a magnification of ZOOx, and the eyepiece line method (Konigsmark, '70; Moatamed, '66) was used in which fluorescent cells are counted as they pass an eyepiece hairline as a result of moving the microscope stage sequentially to cover the entire ganglion section. A cell was considered labeled if its cytoplasm had intensely bright diffuse fluorescence or if distinct fluorescent granules were seen in the cytoplasm. After the fluorescent cells were counted, the coverslips were removed from each section and the sections rehydrated, counterstained with cresyl violet, coverslipped again, and all the cresyl violet stained neurons were counted. The Nissl stained cell counts were made without knowledge of the previous fluorescent cell counts using a Leitz Dialux microscope at 400x magnification. An eyepiece grid reticule (10 x 10 squares) was positioned at the edge of a ganglion and all the Nissl stained cell bodies in the grid counted. Each field was counted within the grid sequentially until the entire section was analyzed and a total cell count obtained for the section (Konigsmark, '70). T o maintain consistency, all counts were done by one individual without knowledge of which animal group was heing counted. T o maintain accuracy, counting of Nissl stained sections was limited to two to four sessions per day, with a maximum of 1 hour per session. Fast blue accumulates within the perikaryal cytoplasm and not within the nucleus (Bentivoglio e t al., '80; Kuypers and Huisman, '84). I t was not possible to differentiate reliably between cells with and without nuclei or nucleoli under fluorescent illumination without experiencing excessive fadi ng of cellular fluorescent label. Therefore all visible neurons with fluorescent label were counted. When each section was recounted for Nissl stained cells, all recognizable neurons were counted regardless of whether nuclei and/or
S.P. LAHR AND D.J. STELZXER nucleoli were visible in the profile and no correction for split cells etc. was made. Cells with very small densely compact nuclei were considered non-neuronal and were not counted in either fluorescent or Nissl material. The percentage of retrogradely labeled fluorescent neurons to the total number of neurons was calculated for each DRG section from each animal and the mean percentage for each ganglia was then computed. The mean percentages from both L4 and 1,s ganglia were summed within an individual experimental group and mean percentages of fast blue labeled neurons were calculated for each experimental group and compared to the appropriate control group. Animals receiving a cervical or a thoracic spinal overhemisection were treated in the same manner.
RESULTS Experiment 1: Tests of dorsal column growth after spinal injury in the newborn rat l.A.l: Degeneration analysis of dorsal column growth. Light microscopic examination of spinal cord sections from control and experimental animals processed for Fink-Heimer impregnation reveals considerable degeneration argyrophilia within the dorsal funiculus and dorsal root projection zones of the lower lumbar and sacral segments. Degeneration argyrophilia is also seen within the dorsomedial portion of the dorsal funiculus through the thoracic and cervical cord in control animals and fills a portion of the gracile nucleus (Fig. 3A,C). In chronic experimental animals degeneration is seen extending through the thoracic cord to within one to two segments of the cervical overhemisection lesion site. Unlike transverse or longitudinal sections examined from comparable levels in control animals (Figs. 2A,C, 4A) the areal extent of the DC projection in the chronic experimental operates diminishes rostrally in thoracic and cervical regions, particularly rostral to Clarke's nucleus, and is apparent in transverse sections only as a small wedge shaped band along the dorso-medial edge of the dorsal funiculus (Figs. 2B,D, 4R).No degeneration staining is seen in any of the experimental animals rostral to the lesion in the cervical cord or in the gracile nucleus (Fig. 3B,D), except for one animal (not shown) which had an incomplete cervical lesion (some of the left dorsal funiculus was uncut). Degeneration argyrophilia is occasionally seen in the vicinity of the lesion but it is not seen within the connective tissue scar a t the border of the lesion or rostral to this level. Artifactual staining due to connective tissue infiltration is seen, but it is clearly different in appearance from axonal degeneration. The connective tissue stains as dark brown intertwining fibrous strands while axonal degeneration argyrophilia is black, punctate, and globular. 1.A.2: A n a l p i s of dorsal column growth b y transganglionic labeling. Bright- and darkfield examination of tissue from normal adult control animals, injected with C-HRP for transganglionic transport, shows a typical dense projection to the left gracile nucleus (Fig. 5A). The topography of'the C-HRP labeled projection in the dorsal funiculus (Fig. 6) is similar to the result of Fink-Heimer impregnation (Fig. 2) except that C-HRP labeled axons, arising only from L4-6 dorsal roots, are positioned more ventrolaterally than fibers arising from the more caudal degenerating dorsal root axons seen with the Fink-Heimer technique. There is no sign of label in the gracile nucleus in chronic experimental
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DORSAL COLUMN AXOTOMY IN NEWBORN RAT SPINAL CORD
Fig. 2. Results of experiment 1.A.1: Fink-Heimer stained sections from upper thoracic spinal levels. A: Low-power photomicrograph of a transverse section from a normal adult control showing th e area containing the most dense DC projection (area between arrowheads) which otiginates from the L5 and caudal dorsal roots. B Low-power photomierograph of a transverse section from a chronic experimental operate a t the
same level as the adult control in A showing the extent of the DC projection (area between arrowheads). Note the marked decrease in size of this projection compared to the adult control. C: High-power magnification of the dorsal portion of the DC projection in (A). D.High-power magnification of the DC projection in (B) indicated by the arrows. Bar (A,B) 100 qm; (C,D) 50 pm.
operates (Fig. 5B). As also seen with Fink-Heimer impregnation in experiment 1.A.1, the topography of C-HRP labeled axons in the dorsal funiculus of chronic experimental operates is similar to normal animals, but i t is obviously diminished in areal extent in thoracic and cervical segments (compare Figs. 2A and 6A with 2D and 6B). Parasagittal frozen sections taken through the cervical spinal cord of chronic experimental operates, processed for
C-HRP, and examined with bright- and darkfield light microscopy, show many labeled axons with thick varicosities within the dorsal funiculus (Fig. 7A). Many of these labeled fibers appear to end as swollen (5-10 pm x 10-30 pm) reactive endings terminating variably over several segments caudal to the lesion (Fig. 8A). No C-HRP labeled axons can be seen growing towards the gray matter in upper thoracic or cervical spinal levels, nor is there any indication of termi-
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Fig. 3. Results of experiment 1.A.1: Fink-Heimer stained sections from the dorso-medial portion of the medulla a t the level of t h e gracile nucleus. 1,ow-power photornicrographofa transversesection from a norma1 adult control (A) and chronic experimental operate (B) showing the presence (A) and absence (€3) of degeneration argyrophilia in the gracile
philia in D contrasted with the dense argyrophilia present in C. Bar (A,R) = 100 Fm; (C,D) = 50 Fm.
nucleus resulting from cutting the L5 and caudal dorsal roots. Highpower magnification of the boxed region of t h e gracile nucleus shown in A and B is shown in C and D, respectively. Note the absence of argyro-
X
P 59
DORSAL COLUMN AXOTOMY IN NEWBORN RAT SPINAL CORD
Fig. 4. Resiilts from experiment. 1.A.1: Fink-Heimer stained, Inngitudinally cut, spinal cord sections. Both A and R are oriented from a dorsal view; top is t,he animal’s right side, bottom is left, right side on the photomicrograph is caudal, and left is rostral. A Photomicrograph of the DC in the cervical spinal cord from adult control material showing the degeneration argyrophilia (outlined by arrows) resulting from cutting the LS and caudal dorsal roots. B: Photomicrograph from the same
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region of a chronic experimental operate showing the extent of the DC projection remaining in the cervical spinal cord near the lesion outlined by arrows. Note the dark fibrous staining of the connective tissue scar (triangular arrowheads), the lack of degeneration argyrophilia in the scar, and the progressively diminished size of the pathway compared to the control (A). Bar = 50 pm.
S.P. LAHR AND D.J. STELZNEK
386
Fig. 5. Results from experiment 1.A.2: C-HRP labeling of transverse sections from dorso-medial portion of medulla a t the level of the gracile nucleus. Low-power darkfield photomicrograph from a normal adult control showing C-HRP labeled axons and terminals in the left gracile
nucleus resulting from a left sciatic nerve injection of C-HRP and transganglionir transport (A) and the lack of labeling in a chronic experimental operate (B). White arrow marks the midline in A and B. Bar = 50
w.
nal labeling in the gray matter a t these levels. There is also no evidence of any damming effect or proliferation of axons or terminals immediately adjacent to the lesion. l.A.3: Ultrastructural analysis of surviuing dorsal column ux0n.s. From a total of 33 distinct cervical and lumbar areas examined in the three chronic experimental animals, electron micrographs of 37 separate reactive endings and axonal varicosities were obtained (Figs. 7C,E, 8B). Many more were observed but were found unsuitable for photographing due to grid bar interference or sectioning and staining artifact. No similar profiles were noted in the similarly processed spinal tissue taken from the same spinal segments in normal control animals. The profiles of endings and varicosities seen in the chronic experimental animals are of similar size and shape to the C-HRP labeled reactive endings and axonal swellings seen with light microscopy described in experiment l.A.2 (see Figs. 7A, 8A). At the electron microscopic level, the axonal varicosities are often found adjacent to nodes of Ranvier (Fig. 7C,E). Both varicosities and enlarged endings are filled with a combination of numerous mitochondria, dense bodies, variously sized granular and agranular vesicles, and a considerable amount of tubular and vesicular profiles of smooth endoplasmic reticulum. Mixed in among the axons are also a few synaptic terminals filled with agranular vesicles that form junctional specializations on isolated dendrites (not shown). These synaptic knobs are also apparent in small numbers in control material. The enlarged reactive endings did not appear to make synaptic contacts in the dorsal column although none were serially sectioned to confirm this point. The reactive ending shown in Figure 8C does have an infolding of plasma membrane with a potential junctional specialization and was the only profile suggestive of this possibility.
of newborn animals injected with C-HRP shows label present ipsilaterally in the left gracile nucleus by 42 hours after birth (Fig. 9). The labeling pattern in the rostra1 portion of the gracile fasciculus and the entire gracile nucleus in newborn animals is grossly similar in topography to the labeling seen in normal adult animals (compare with Fig. 5A), although the neonatal material is less densely labeled. 2.B: Retrograde labeling of lumbar DRG neurons. Cell counts show the same percentage of neurons retrogradely labeled in animals injected as newborns or adults. An average of approximately twelve percent of the total number of L4 and L5 DRG cells in each section sampled are retrogradely labeled in each group (Table 1).However, considerable variability is noted comparing different animals and ganglia from each group (Table 3). As many as 20”( of DRG cells are labeled in the L4 DRG of newborn rase 102.1, while as few as 6% are labeled in the L4 DRG of case 102.9. Similarly, 19% of the DRG in the L4 DRG of adult case I 14.4 are labeled but only 7 % of the L5 DRG of case 114.6 are labeled. Although our method of analysis does not allow a valid estimate of the total number of cells in each DRG sampled, it is likely from our data of the average number of cells labeled in each section and of the average total number of cells counted in each of these sections that we are dealing with a similar population of cells in both age groups (Table 4). A similar number of labeled (31 2 7 and 38 5 for newborn and adult groups, respectively) and unlabeled (273 98 and 296 fr 81) cells are seen. Labeled cells are found variably throughout a ganglion in both newborn and adult animals and no consistent pattern of localization within a ganglion is observed (Figs. 10 and 11).Cell measurements were not made but the labeled cells are from the medium- and large-sized neuronal populations a t both ages.
Experiment 2: Tests of dorsal column maturity in the neonatal rat
Experiment 3: Test of DRG cell survival after neonatal spinal lesion
2.A: Transganglionic labeling. Light microscopic examination using both bright- and darkfield illumination
The percentage of L4 and L5 DRG cells labeled in individual sections from newborn animals receiving medulla
DORSAL COLUMN AXOTOMY IN NEWBORN RAT SPINAL CORD
387
Fig. 6. Results from experiment 1.A.2: C-HRP labeling of transverse sections from upper thoracic spinal cord showing topography and size of the C-HRP labeled DC pathway arising from a left sciatic nerve injection, i.e., L4-6 DRG. Darkfield photomicrograph from an adult control
(A) and a chronic experimental operate (B) of the left dorso-medial portion of the spinal cord a t the same segmental level. Note t h e markedly decreased extent of the labeled DC pathway in the chronic experimental operate (B) compared to the control (A). Bar - 50 ym.
TABLE 1. Summary of Results From Experiment 2.B'
TABLE 2. Summarv of Results From Experiment3'
Procedilre
Agehterval
Code no.
-
n
X% (U&5DRG)
-
X%
Code
S.D.
Procedure
&elinterval
no.
n
(L4&5DRG)
S.D.
(106)
6
9
4
(1W)
5
10
3
(105) (115)
6
10
3
(104)
4
10
7
~~
FB injection SllCIifioe FB injection SaCIifiCe
PD 0 PD 2 adult + 3 dam
(102) (114)
6 6
12 13
5 4
'Shown are the mean percentages (XW)and standard deviations (S.D.) of retrogredely labeled neurona per section computed from combined IA and L5 DRG counts for all animals rsceiving an mjection of fast blue (FB)as newhorn (PD 0) (mde 102) or adults (code 114) and having 2 and 3 day patinjection periods. respectively.See Methods and section 2.B fordetails.
FB injectinn Overhemisection SaCXifiOe FR injection Sacrifice FB injection Overhemisection SaCrifiCe FB injection Sacrifice
PD 0 PD 3 PD 10 PD 0 P n 10 PD 0 PD 3 PD 22 PD 0 PD 22
'Shown are the mean percentages (XW) and standard deviations (S.D.) of retrogradely labeled neurons per section computed from combined I 4 and L5 DRC counts for all animals receiving an injection of fast blue (FB)as newborn (PD 0) and having 10 day postinjection survival p e r i d with a lesion (code 106), or unlesioned (code 107), and 22 day postinjection survival periods with a lesion (code 105, 115). or unlesioned (code 104). See Methods and section 3 for details.
389
DORSAL COLUMN AXOTOMY IN NEWBORN RAT SPINAL CORD injections of fast blue decreases slightly to approximately lor'(in all groups of rats with either 10 or 22 day postoperative survival periods (Table 2). The likely reason for this difference compared with the newborn group described in experiment 2.B, above, is that retrogradely labeled neurons generally have fainter fast blue fluorescence and fewer granules dispersed in the cytoplasm compared to animals with the shorter 2 day survival period given in experiment 2.B. A small number of poorly labeled neurons counted in experiment 2.B may have been missed in the present experiment. Labeled cells are again found scattered variably throughout a ganglion in both experimental and control animals and no consistent pattern of localization within a ganglion is observed (Fig. 12). Cell measurements were not made but, as in experiment 2.B, the labeled cells are from the medium- and large-sized neuronal populations at both postinjection survival times. There is no clear effect of either a high cervical or midthoracic overhemisection spinal injury made at 3 days of age. Control and experimental groups have the same percentage of cells per section labeled, and there is similar variability between groups of this experiment to that seen in experiment 2.R. For instance, after a 10 day survival, over 1 4 ' ~of the L4 ganglion cells are labeled in control animal 107.1, but only 6'c are labeled in the L5 DRG of control animal 107.5 (Table 3 ) . A similar variabilily is seen in animals receiving an overhemisection injury at 3 days of age. Over 16% of the ganglion cells are labeled in the L4 DRG of experimental animal 106.1, while only 5'0 of the ganglion cells are labeled in the L5 DRG of experimental animal 106.4 (Table 3). After a 22 day survival period, the L4 DRG of control animal 104.5 has over 25"< of its cells labeled, the highest percentage of all groups, while the L5 DRG of 104.7 has less than 4 % laheled (Table 3). No real difference is seen if animals with this survival time have an overhemisection injury at 3 days of age. For instance, 13r, of the L5 DRG of experimental animal 105 1 are labeled, while less than 600 of the cells in Ihe L5 DRG of experimental animal 105.5 are labeled (Table 3 ) . There is also no clear difference between animals with or without the spinal overhemisection injury when the mean number of cells counted per section and the mean number of these cells labeled with fast blue per section are compared between groups (Table 4). Clearly, the overlap in the standard deviations between groups would not lead to statistical significance and numbers fall within a similar range. However, the average number of cells per section and average numbcr of labeled cells per section ranges between 18"~and 2 5 " ~less when groups receiving overhemisection injury are compared with the control groups.
DISCUSSION Dorsal column growth after neonatal spinal injury These experiments show that ascending DC axons, which lie adjacent to descending CST axons in the rat, do not exhibit the same ability as CST axons to grow around a spinal cord overhemisection lesion made at birth (experiment 1 ). This lesion severs the dorsal funiculus bilaterally, which is the region in the rat spinal cord normally containing both TABLE 3. Results From Exueriments 2.B.and 3' -
Procedure
Amdinterval
Animal codeno.
FB injection Sacrifice
PD 0 PD 2
-
X%
S.D. 5.78 5.56 5.07 3.18 4.04 2.19 1.75 1.99
adult + 3d a y
PD 10
10.43 7.86
3.50
13
7 7 8 8 10 10 11 11
8.16 11.37 25.49 8.97 13.44 10.36 5.79 3.63
2.50 6.70 6.50 3.67 3.79
(102.3)
Id
I
L5
6 6 6 10 10 5
(102.lO)
L5 L4
(114.1)
L5 L4 L5 Id L5 Id L5
5 6 6 9
(114.4)
L4
(114.5)
L5 Id
11 10 6 6 6 6 8 7 8 8 6 6 7 7 10
(106.1)
L5 Id 5 I. Id
L5 (106.3)
LA Id
L5 (106.4)
Sacrifice FB injection Sacrifice
PD 10 PD 0 PD 10
IA L5
(106.5)
LA
(106.6)
L5 L4 L5
(107.1)
Id
(Irn.2) (107.3)
(107.4)
(107.5)
L5 L5
L4 L5 Id L5
L4
L5 FB injection Thoracic overhemisedion
PD 0
(105.1 )
Id
PD 3
(105.2)
L4 L5
Sacrifice
PD 22
(106.3)
Id L5 Id L5
FB injection Cervical overhemisection SaCIifiCe FB injection Sacrifice
5 6
L4
(106.2)
PD 3
6
Id
L5
Thoracic overhemisection
I
L5 (102.9)
(114.6)
SfIcrifie.2
L4 L5
L5 L4 L5
7
(102.2)
(114.3)
PD 0 PD 3
7 12 12 13
I
(114.2)
FB injection Cervical overhemisection
L5
L4
(102.8)
FB injection sacrifice
19.63 14.41 18.48 9.93 12.75 19.7 14.1 10.65 5.63 11.12 7.26 8.12 15.7 10.62 16.85 9.88 16.5 9.96 19.04 10.14 15.7 9.75 14.38 7.22 16.45 8.84 14.33 6.55 7.35 6.0 6.45 4.55 8.67 6.43 11.25 12.35 14.16 12.77 13.11 11.83 9.77 8.28 9.09 7.2 5.55 7.43 13.05 10.06 6.53 10.03 9.87 10.27 11.4 8.85 5.81
(102.1)
L5
(106.5)
~
No. of sections ~
(105.4)
Fig. 7. A Brightfield photomicrograph of a parasagittal section through the lower cervical spinal cord showing C-HRP labeled axons from a chronic experimental operate. Note numerous varicosities and axonal enlargements with some constrictions shown a t arrows. B-E: Electron micrographs of longitudinally cut cervical (C) and lumbar (E) spinal cord from chronic experimental operates (without C-HRP injection). The axonal enlargements shown in (C and E) are filled with numerous cytoplasmic organelles including: mitochondria, dense bodies, vesicles of various sizes, and profiles of smooth endoplasmic reticulum. Note axonal expansions formed adjacent to nodes of Ranvier (arrowheads). D: 'I'ransversely and obliquely (B) cut profiles of organelle filled axonal enlargements. Note unusual pattern of smooth endoplasmic reticulum in B. Bar (A) 50 pm; (B-E) = 0.5 pm.
Ganglia
L4
9
10 10 7 5 10 10 10 10 10 10 10 10
5 6 5 6 5 I
5
1.45 1.21 3.99 3.42 2.34 5.09 2.68 2.35 4.52 2.31 3.98 3.38 1.84 1.49 2.28 2.78 3.4 2.7 4.2 2.62 1.8 1.94 1.85 1.46 2.59 1.25 2.59 3.49 0.92 4.99 4.58 1.72 2.12 1.56 2.24 2.06
1.20 3.43 3.08 1.94 1.49 3.67 3.24 1.67 3.04 3.43 2.21
PD 0 PD 3 PD 22 PD 0 PD 22
(115.1) (104.1) (104.5)
IA L5 Id L5
(104.6)
IA L5
(104.7)
L4 L5
2.14
1.84 0.56 1.48
'Shown are the mean percentages (XLZ)and standard deviations (S.D.) of fast blue (FB) labeled n e u r o n s c a l c u l a t e d f r ~ m ~ I 2 s e ~ ~ f o r e a c h l r description. This table ia summarized in Table 1 (experiment 2.B.)and Tahle 2 (erperiment 3).
390
Fig. 8. A: Brightfield photomicrograph of a parasagittal section through the lower cervical spinal cord several segments caudal to the lesion site showing a C-HRP labeled reactive ending in a chronic experimental operate. B: Electron micrograph of a similiar organelle filled enlarged reactive ending as seen in A but not injected with C-HRP. Th e ending is marked by arrows and the unmyelinated axon extends past the right edge of the electron micrograph. C Enlargement of the reactive
S.P. LAHR AND D.J. STELZNER
ending shown in B bordered by arrows. Note the large number of agranular and granular vesicles that are prominent in reactive endings. Also note the possibility of a synaptic junctional specialization formed with an unidentified profile (arrow). This is the only example of a potential synaptic cnntact formed hy a reactive ending t h at we have observed in our analysis. Bar (A) = 50 pm: (B,C) = 0.5 Fm.
391
DORSAL COLUMN AXOTOMY IN NEWBORN RAT SPINAL CORD TABLE 4. Summary of Cell Counts' -
Animal
Procedure FB injection Sacrifice FB injection Sacrifice FB injection Overhemiseaion Sncrifioe FB injection Sacrifice FB injection Overhemisection Sacriiim FB injection Sacrifice
X no. of FB
-
No. of sections
(IA & 5 DRG)
S.D.
WllS
PD 0 PD 2
79
213
98
31
Adult
a5
296
81
38
92
259
87
23
85
316
108
29
e6
221
102
21
72
294
92
28
Agehnterval
code no.
X no. of c e h
S.D.
+3dnya
PD 0 PD 3 PD 10 PD 0 PD 10 PD 0 PD 3 PD 22 PD 0 PD 22
'Shown are the mean numben (X) of Nissl stained DRG cells counted per section ("Cells") and fast blue retrogradelylabeled DRG celk per section ("FBCells"),from both Ld and L.5 ganglia combined, and standard deviations(SD.)for each group in experimenla 2.8 and 3.
els and is similar in density and distribution when chronic experimental operates are compared with adult controls using Fink-Heimer or C-HRP techniques. Both techniques also demonstrate a progressive diminution of labeled DC axons a t more rostra1 spinal levels in chronic experimental operates as the lesion was approached. Thus it is likely that our results represent a failure of DC axons to grow around the lesion. The lack of growth by DC axons in animals during the period CST axons continue to elongate around the same lesion, could be due to several different factors:
Matrix factors
Fig. 9. Results of experiment 2.A: C-HRP labelling of the neonatal DC projection in the gracile nucleus. This low-power darkfield photomicrograph shows considerable C-HRP label, indicating that DC axons are present in the ipsilateral gracile nucleus within 36 hours after birth. Compare with the adult pattern shown in (Fig. 5A). Bar 50 wm. =
pathways. These negative findings are unlikely to be due to methodological deficiencies, since two very different and reliable tract tracing techniques were used. The Fink-Heimer technique, the first method used in these experiments, has been used by many investigators to demonstrate developing axons (Schneider, '68, '73; Anker and Cragg, '74; Hicks and D'Amato, '75) and to demonstrate axonal sprouting in adult animals (Liu and Chambers, '58; Goodman and Horel, '66). This method is especially useful for studying axonal growth in the adult animal after early brain injury (Prendergast and Stelzner, '76a; Stelzner et al., '79), since degeneration is cleared very rapidly after neonatal injury (Gilbert and Stelzner, '79). The C-HRP conjugate was also used for transganglionic tracing in these studies because it labels axons via the axonal transport system and is more sensitive than wheat germ agglutinin-horseradish peroxidase conjugate (U'GA-HRP) as a t,ransganglionic probe of myelinated axons (Wan et al., '82; Trojanowski et al., '81; Trojanowski, '83). The dorsal root projection is densely labeled in transverse sections at lumbar spinal lev-
Since CST axons are able to continue elongating after spinal overhemisection injury during the early postnatal period (Bernstein and Stelzner, '83; Schreyer and Jones, '83), the CNS environment used by these axons is permissive for axonal growth. The spinal cord environment is quite different during this period than after maturation is complete. Most gliogenesis occurs during the early postnatal period (Gilmore, '71) and immature astroglia appear to express substances (e.g., laminin, nerve cell adhesion molecules, etc.) that support axon elongation (Silver et al., '82; Liesi et al., '83; Silver and Rutishauser, '84). Myelination of the spinal cord also occurs postnatally (Matthews and Duncan, '7i). Therefore, factors that appear to be present in mature astrocytes (Liesi et al., '83; Silver and Ogawa, '83; Smith et al., '86) and oligodendrocytes (Schwab and Thoenen, '85; Schwab and Caroni, '88), which inhibit axonal growth, are probably not present in the early postnatal period. Bregman has shown that neonatally injured spinal serotonergic (Bregman, '87) and rubrospinal (Bregman and Reier, '86) axons can regrow through embryonic spinal tissue implanted into a newborn transection site, and continue elongating caudally in the neonatal rat spinal cord. All of these features indicate that the neonatal rat spinal cord is relatively growth permissive. The matrix remaining in the unlesioned portion of spinal cord in our study should be essentially the same for both CST and DC axons during the early postnatal period. It is possible that there are additional environmental factors a t work such as the cephalo-caudal gradient of maturation seen in the spinal cord that might interfere with ascending axonal elongation in the developing spinal cord. Dodd and ,Jessell ('88) reviewed several recent studies which support the concept that several molecular mechanisms may exist which allow axons to select pathways by recogniz-
392
S.P. LAHR AND D.J. STELZNER
Fig. 10. Fluorescent photomicrograph of a typical DRG section from a newborn rat injected with fast blue and given a two day transport period. Cell counts reveal (see Table 1) approximately 124. of the neurons are labeled. Examples of labeled cells are indicated by arrows. Compare with Figures 11 and 12. Bar = 100 Gm.
ing specific cues in their environment and that the nature of these cues change along both temporal and spatial gradients during development. Nevertheless, the maturation of CNS matrix does not appear to be the chief factor limiting axonal elongation since CST axons are able to grow around either a high cervical or a midthoracic lesion site until the same postnatal age (postnatal day 6, Bernstein and Stelzner, '83; Bates et al., '88). This growth occurs even though the cervical spinal environment is relatively more mature at this time than thoracic cord. In addition, following a spinal overhemisection injury, few CST axons return to the dorsal funiculus pathway caudal to the injury even though this area should be the most appropriate pathway for CST growth and is the most immature region of the spinal cord white matter at caudal spinal levels.
Retrograde cell death Neuronal survival after axotomy is critical to axonal regrowth. Several studies have demonstrated considerable cell loss in lumbar DRGs following sciatic nerve lesions in kittens (Riding et al., '80; Aldskogius and Riding, '81) and neonatal rats (Ranson, '09; Bondok and Sansone, '84; Schmalbruch, '87). Dorsal root ganglion cell loss has also been reported in neonatal rats following a dorsal root lesion (Yip et al., '84). This "Gudden effect" (see Brodal, '40; and Lieberman, '71) has also been shown for some descending pathways in kittens (Bregman and Goldberger, '83) and rats (Prendergast and Stelzner, '76b; Goshgarian et al., '83; Bregman and Reier, '86), but is not always the case since many spinal ascending projection neurons (Rryz-Gornia and
Fig. 11. Fluorescent photomicrograph of typical DRG section from an adult rat injected with fast hlue and given a 3 day transport period. Cell counts indicate (see Table 1) approximately 1 2 4 of the neurons are
labeled. Compare with Figures 10 and 12. Examples of labeled cells are indicated by arrows. Bar = I00 wm.
394
S.P. LAHR AND D.J. STELZNER
Fig. 12. Fluorescent photomicrographs of typical DRG sections from animals injected a t birth with fast blue and given a 10 day postinjection survival period. A: Control animal not receiving spinal injury. B: Experimental operate receiving a cervical spinal overbemisection lesion a t 3 days of age. Examples of labeled cells are indicated by arrows. Note
the relative similarity in number of labeled neurons and variable distribution in both cases. Cell counts reveal (see Table 2) t h a t about 9-10? of'the cells are labeled in both lesioned and unlesioned groups a t both 10 and 22 day postinjection survival periods. Bar = 100 pm.
Stelzner, '86) and descending corticospinal tract neurons (Rates and Stelzner, '87) survive neonatal axotomy. In our studies (experiment 3), prelabeling DRG neurons and comparing the percentage of labeled cells between neonatal operates and controls, little sign of DRG cell death is seen following a DC injury. Both experimental and control operates have approximately 10% of the L4 and L5 DRG neurons labeled with fast blue (see Table 2), and no difference is seen in the percentage of prelabeled DRG neurons of neonatal operates if the lesion is made a t a thoracic or a cervical level. The slight decrease in percentage of labeled neurons (from 1200 to 10 % ) from normal newborn and adult cases is attributed to decreased fluorescence seen a t the longer 10 and 22 day survival periods used in the present analysis. Previous work has shown that most retrograde changes and neuronal death following neonatal axonal injury occur very quickly and is complete by 6 to 7 days postoperatively (Romanes, '46; LaVelle and LaVelle, '58; Yip et al., '84; Bregman, '88j, so both survival periods are after the period that cell loss should have occurred. The fast blue injection site and amount of fast blue retrogradely transported to label DRG neurons is likely to vary somewhat from animal to animal (Kuypers and Huisman, '84). Since fluorescent label tends to fade during illumina-
tion, cell percentages were gathered from all cells labeled on each section, rather than identifying and counting only cells with visible nuclei. Sections sampled in our analysis were taken a t uniform intervals throughout each ganglion, but we were unable to standardize totally the number of sections analyzed and the exact region of the DRG sampled. These variables are likely to be responsible for the size of the standard deviations for each group (in the range of 3-790). Cell counts of fluorescently labeled or Nissl stained DRG neurons further suggest that relatively little cell loss occurs (Table 4). Again, similar mean averages of each parameter are seen and there is considerable overlap in the standard deviations between groups. However, especially for the cell count data, the large standard deviations could cover a small cell loss. Since other estimates of the percentage 01lumbar DRG cells projecting to the gracile nucleus vary between 10-15'% and 20-25"; (Fyffe, '83; Geisler et al., '84;Richardson and h a , '84), cell loss in our study remains a possibility. Even though there is no difference in the percentage of DRG neurons labeled per section in groups with or without spinal overhemisection injury, there is a trend for lesioned groups to have fewer cells in each section of the DRG sampled and fewer labeled cells per section. This point could have significance if the fast blue injections failed to prelabel a propor-
395
DORSAL COLUMN AXOTOMY IN NEWBORN RAT SPINAL CORD tion of the DRG neurons projecting to the gracile nucleus prior to axotomy, and a proportion of both labeled and unlabeled cells underwent post-axotomy retrograde cell loss. The possibility of a small cell loss will have to be tested using other methods of analysis which give less variability between samples. However, even if some cell loss occurs, our data show that many neonatal DRG cells axotomized by spinal overhemisection injury survive this injury at the postoperative periods tested and maintain axons within the dorsal funiculus.
Regenerative sprouting Other experiments have suggested that formation of inappropriate synapses by cut axons removes the stimulus for regenerative growth (Bernstein and Bernstein, '67, '69, '71). Our results do not support this hypothesis. We see no indication of axon growth into the gray matter caudal to the lesion in our Fink-Heimer stained or C-HRP labeled material. The nature of the reactive endings in our experiments with chronic experimental operates after newborn injury is still unclear, but there does not appear to be proliferation of cut axons in the vicinity of the lesion, a t least at the p.0. periods tested. Although other investigations using shorter survival periods after axonal injury in adult animals (Ram6n y Cajal, '28: Brown and McCouch, '47). showed extensive axonal growth into or near CNS injury, such growth was abortive and decreased with time. Our C-HRP filled endings in chronic operates were found a number of segments away from the lesion site and resemble the silver stained terminal clubs described by Cajal as abortive regeneration in adult lesioned animals (Ram6n y Cajal, '28). The axonal varicosities and enlarged reactive endings we see in our material at the electron microscopic level also appear similar to the H R P labeled axons we see at the light microscopic level and to reactive endings after axonal injury in the adult CNS (Lampert and Cressman, '64; Gilson and Stensaas, '74). These findings suggest a progressive retrograde retraction process by the injured axons and are in agreement with the slow retraction of corticospinal tract axons seen after pyramidal lesions in adult hamsters (Kalil and Schneider, '75) and mice (Fishman and Kelly, '84).
Collateral sprouting Another possibility for the lack of dorsal column regeneration is that segmental collaterals sprout either as a result of denervation of synaptic sites in the lumbar spinal cord caudal to the spinal injury, or in response to the loss of connections of the DRG cells in the gracile nucleus due to axotomy ("pruning"). There are a number of studies indicating dorsal root sprouting occurs in adult and developing animals (Liu and Chambers, '58; Murray and Goldberger, '74; Stelzner e t al., '79) as well as studies showing axonal sprouting of other spinal pathways (Prendergast and Stelzner, '76a; Prendergast and Misantone, '80). However, it is not known if sprouting by dorsal column afferents occurs in the lumbar spinal cord after the high cervical overhemisection lesion made in the present study. If Schneider's concept of "pruning" (Schneider et al., '85) is correct and there is the need or ability of' a neuron to maintain a certain amount of arborization and synapse formation, then cutting DC axons should result in an increased growth of segmental collaterals remaining caudal to the spinal injury (Schneider, '73; Pickel et al., '74; Schneider and Jhaveri, '74). Such segmental sprouting might result in a decreased regenerative response
and may be one factor related to the loss and apparent retract,ion of DC collaterals.
Regenerative signals Oblinger and Lasek ('88) recently presented evidence showing different rates of selected protein transport for DRG cells depending on whether the peripheral or central process (dorsal root) was damaged. Following peripheral nerve crush, smaller than normal ratios of neurofilament protein/tuhulin are transported in the peripheral process, while the transport of neurofilament protein and tubulin is not changed in the central process. In contrast to this, when the dorsal root is crushed, there is no detectable change in the transport of neurofilament protein or tubulin in central or peripheral branches. Similar results were shown previously by Perry and Wilson ('81) and Hall ('82). Most morphological studies also show chomatolysis after injury to the peripheral but not the central DRG process (reviewed by Cragg, '70; and Lieberman, '71). Because of this differential effect caused by central or peripheral axotomy on the DRG neuron, Oblinger and 1,asek ('88) concluded that the cytoskeletal networks are affected differently and speculated that the injury to the dorsal root produces a suboptimal signal to initiate metabolic changes in DRG cells related to axonal regeneration. The effect of injury to the DC axons on protein transport, is unknown in the newborn rat, but it is likely to be similar to Oblinger and Lasek's ('88) findings since the elongation of this pathway is complete at birth (see previous discussion). Oblinger and Lasek's work also adds support to the previous findings of Richardson and Issa ('84), who showed that regenerative growth of DC axons into peripheral nerve grafts following DC injury in adult rats is enhanced when thc peripheral process of these neurons is injured within one week of graft implantation. Richardson and Verge ('87) later showed that the rate of growth of regenerating dorsal root axons is two to three times greater when the peripheral process o r the DRG is also damaged. Although this growth enhancement effect differs from the priming effect on peripheral nerve regeneration by a preceding crush injury (McQuarrie and Grafstein, '73; McQuarrie et al., '77), the efTect may work by a similar mechanism since the metabolic machinery related t o elongation is enhanced with either type of injury.
Maturation The C-HRP labeling of DC nuclei in newborn rats (experiment 2.A) and the retrograde labeling of the same percentage of DRG neurons in newborn and adult animals (experiment 2.B), indicate that a substantial portion, if not all, of the DC pathway has grown through the cervical cord at birth. Our experiments also corroborate the work of Chimelli and Scaravili ('87), who studied the development of the DC in fetal rats with electron microscopy and wheat germ agglutinin-horseradish peroxidase transport techniques. Our results and theirs indicate that a spinal overhemisection a t C2 in newborn rats severs ascending DC axons which have already terminated in the dorsal column nuclei. Most CST axons in the neonatal rat are uncut and still elongating when a spinal overhemisection is made a t birth, which interrupts their normal pathway (Bernstein and Stelzner, '83; Schreyer and Jones, '82, '83). It appears that some aspect related to the different maturity of these two classes of neurons occupying anatomically adjacent
S.P. LAHR AND D.J. STELZNER
396 pathways may be the major factor responsible for the different axonal response to the same spinal cord injury. Target recognition by growing axons followed by the initiation of synaptogenesis are both factors that regulate axonal elongation. The CST elongates through the length of the spinal cord during the first 9 to 12 postnatal days (Donatelle, '77; Schreyer and Jones, '82, '88a,b) and synaptogenesis appears complete by the end of the second postnatal week, at least in the lumbar intermediate gray (Weber and Stelzner, '80). Recent experiments have shown that. CST growth in the rat, occurs around a high cervical spinal overhemisection made during the first postnatal week but that the extent of growth is greatly reduced between 6 and 1 2 days of age (Bates e t al., '88; Weber et al., unpublished findings). This aberrant growth, a t least, in postnatal day 6 operates, is probably from cut fibers, although a population of late growing CST axons has not been ruled out (see Tolbert and Der, '87). When an elongating axon reaches its target the neuron changes from a growth state, producing membrane, cytoskeleton, and other materials necessary for elongation, into a conductive state that emphasizes synaptic function (see Skene, '84). The protein GAP-43 is one of a group of proteins transiently expressed and anterogradely transported in temporal correlation with P N S and CNS axonal development (Skene and Willard, '81a,b; Benowitz et al., '81; reviewed in Skene, '84; and Benowitz and Routenberg, '87). This growth-associated protein is found a t low levels in adult PNS and CNS, and the amount of synthesis and anterograde t,ransport has been shown to increase markedly after injury to axons which successfully regenerate in the PNS, while a similar increase is not seen after injury to CNS axons (Skene and Willard, '81b). Meiri et al. ('86, '88) have localized GAP-43 in axonal growth cones and neurites and this result combined with the differences in GAP-43 in regenerating and nonregenerating axons suggests an association of GAP-43 with axonal elongation. Kalil and Reh ('79, '82) have shown evidence for regenerative growth of cortical axons in an aberrant pathway following pyramidotomy in the early postnatal hamster. A recent study by Kalil and Skene ('86) has shown an elevated synthesis and transport of GAP-43, which is temporally correlated with the growth of the pyramidal tract in the postnatal hamster. The time course of this elevation is not altered hy neonatal pyramidotomy. Lesions of the pyramidal tract in adult hamsters also do not reinduce an increase in synthesis and transport of GAP-43. Of interest to our experiments would be to determine the amount of synthesis, transport, and localization of G.4P-43 in the postnatal rat comparing the CST and DC pathways during development and after neonatal spinal injury. It may be that damage to DC axons does not result in the re-expression of GAP-43 synthesis (see Willard and Skene, '82), while GAP-43 synthesis and transport continues in the elongating CST after axotomy. The exact interaction of neuronal and environmental influences on CNS axonal regeneration is still not fully elucidated, but our results support a significant role for the interaction of neuron and environment related to axonal maturation. The particular aspect of maturation we believe is key involves the cessation of axonal elongation when a target has been reached and when synaptogeneis is initiated. Although it is likely that there are differences in the maturation of local non-neuronal elements within the dorsal funiculus, t,he adjacent CST and DC axons should have access to the same factors in the unlesioned spinal tissue. It may be
that environmental growth cues or factors specific for DC axons arc gone, or are at very low levels, in the neonatal rat or that local cues or factors affecting the CST no longer have an effect on the more mature DC system. I t would be interesting to determine if re-expression of proteins related to axonal elongation could be induced in dorsal column axons and would result in extensive axonal elongation within the postnatal rat spinal cord.
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