Proc. Natl. Acad. Sci. USA Vol. 81, pp. 6245-6249, October 1984 Neurobiology Developing dorsal root ganglion neurons require trophic support from their central processes: Evidence for a role of retrogradely transported nerve growth factor from the central nervous system to the periphery (dorsal root axons/sensory neurons/retrograde transport/trophism) HENRY K. YIP AND EUGENE M. JOHNSON, JR. Department of Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110 Communicated by Oliver H. Lowry, June 11, 1984 ABSTRACT Injury to the peripheral processes produces a response." The lesser role of the central relative to the pe- profound cell loss (40-50%) in the dorsal root ganglion ofnew- ripheral process could be due, in part, to the disproportion- born rats. Although division of central processes produces lit- ate loss of axoplasmic volume between the central and pe- tie or no cellular change in sensory ganglion of adult animals, ripheral axons after axotomy because the central axons are no Information has been available on the effect of dorsal root of smaller diameter (7) and the flow of axoplasm is slower section in developing dorsal root ganglion. We show that 6 than in the large peripherally directed axons (8, 9). days after dorsal rhizotomy on newborn rats, there is a 50% Populations of developing neurons come under the influ- decrease in neuronal number in L5 dorsal root ganglion. A ence of their peripheral fields and are able to make compen- combined central and peripheral lesion of the sensory process satory adjustments according to the sizes of the afferent in- results in a greater decrease in neuronal number (70%). Both put they receive (10-12) and the efferent output they project of these effects can be prevented by the concomitant treatment (13-16). The adjustments are made by way of corresponding with nerve growth factor. We also demonstrate that 12'I-la- alterations in cell number and size within the developing beled nerve growth factor is retrogradely transported with neuronal population. The sensory ganglion is unique in the high selectivity from the spinal cord to the dorsal root ganglion sense that its target areas include both central neurons in the via the dorsal roots. The results indicate that trophic support spinal cord and the innervated organs in the periphery. De- for developing sensory neurons is provided through the central spite the lack of morphological alteration reported after sec- processes. This is presumably due to the uptake and retro- tioning of the central process in adult animals, it is possible grade transport of a trophic factor by the terminals of the cen- that developing DRG neurons receive trophic support from tral processes. The data suggest that nerve growth factor may both central and peripheral target zones. be the trophic factor. Nerve growth factor (NGF) has long been thought to act as a trophic factor transferring information from the periph- Dorsal root ganglion (DRG) neurons, developing as bipolar eral target organs to the innervating sympathetic and sensory neurons from the neural crest, are known to have two very neurons. Our recent findings (17) have demonstrated that similar processes. Both the central and peripheral processes eliminating the peripheral target influence by crushing the have structural characteristics of axons (1, 2). The axons of sciatic nerve in the newborn rat resulted in a substantial loss the spinal sensory neurons bifurcate in the DRG, sending (40-50%) of neurons in L5 DRG. Treatment with NGF pre- one branch in the dorsal root centrally toward the spinal cord vented the decrease in neuronal number of the axotomized and the other in the spinal nerve toward the peripheral tis- DRG. In the present study, we examine the effects ofcentral sues. Axonal injury of the peripheral branch in adult animals process axotomy on the survival of the neonatal neurons to leads to a profound reaction in the DRG neurons. In con- evaluate the relative roles of central and peripheral contacts trast, section of the dorsal roots proximal to the DRG in on the development of the DRG neurons and to determine if adult animals does not initiate significant morphological exogenous NGF can prevent the observed deleterious ef- change in the ganglion cells (3-5). fects of central axonal injury. One mechanism postulated to initiate neuronal response to It is generally accepted that the physiological effects of injury is that neurons depend on trophic substances coming NGF on target neurons are mediated via retrograde trans- from target cells for their survival and normal maintenance. port of NGF from the target to the neuronal cell body (18). If Interruption of the supply of the trophic substances would NGF were a factor involved in providing trophic support for then initiate the response (6). A reasonable explanation for DRG neurons through the central process, it would be pre- the nonresponsiveness of DRG neurons to sectioning of dor- dicted that 125I-labeled NGF (125I-NGF) should be specifical- sal roots would be that the central process plays no role in ly retrogradely transported from the terminal fields of senso- the trophic support of DRG neurons or, alternatively, that ry neurons in the spinal cord to the DRG neurons. The data the loss of a postsynaptically derived trophic substance from presented here document that 1251-NGF is so transported. the central terminal zones can be sufficiently replaced by trophic agents from the peripheral target cells to keep the MATERIALS AND METHODS neurons adequately maintained. In the latter case it would be Experimental Animals. Litters of newborn, postnatal day assumed that the loss of supply of trophic substance by the (PND)-0 (-6.5 g), and adult (250-300 g) Sprague-Dawley centrally directed axons in the dorsal roots was quantitative- rats (Chappel, St. Louis, MO) of either sex were used in this ly relatively small and thus the cell does not show an "injury study. They were kept in controlled temperature (230C + 2°C) and in a light/dark cycle of 14/10 hr with food and water The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: NGF, nerve growth factor; DRG, dorsal root gangli- in accordance with 18 U.S.C. §1734 solely to indicate this fact. on; PND, postnatal day. 6245 Downloaded by guest on September 30, 2021 6246 Neurobiology: Yip and Johnson Proc. NatL Acad Sci. USA 81 (1984) ad libitum. Litter sizes were adjusted so that each had be- with isotonic saline followed by perfusion with 10% formal- tween 8 and 10 pups. in. The L5 DRG were dissected, dehydrated with ethanol, Surgical Procedures. The two operative procedures were and embedded in paraffin. Complete serial sections of 8 pim dorsal root section and sciatic nerve crush. For either proce- thickness were cut and stained with 0.1% toluidine blue. The dure, newborn rats were anesthetized by cooling and adult contralateral L5 DRG served as control for the axotomized animals were anesthetized with an intraperitoneal injection DRG. To estimate the total number of neurons in the L5 of chloral hydrate (350 mg/kg). For dorsal root section, a DRG, cells in every 10th section of each ganglion were laminectomy was performed at the level of T13 and L1 verte- counted at a magnification of 400x on coded slides. Only brae, exposing the L4 and L5 segments of the spinal cord. cells with at least one prominent nucleolus in the plane of The 5th lumbar (L5) dorsal roots were transected at the point section were counted. Viable neurons, containing prominent of entry into the spinal cord on the right side of the animal. Nissl substance and a large nucleus with one or more nucleo- The exposed spinal cord was covered with Gelfoam; the li, were easily identified. The correction factor for split nu- muscles and the skin were sutured. The right sciatic nerve cleoli was calculated according to Konigsmark (23). The sta- was exposed in midthigh and crushed for 40 sec with a pair of tistical significance of differences obtained among treated jeweler's forceps at the tendon of obturator internus. In the and untreated animals was evaluated by using a two-way sham-operated animals, both the dorsal root and the sciatic analysis of variance with Satterwaithe's correction of de- nerve were exposed but not lesioned. grees of freedom due to unequal group size (24). Preparation and Labeling of NGF. The 2.5S mouse NGF was prepared from submaxillary glands of adult male mice as RESULTS described by Bocchini and Angeletti (19). NGF solution was Effects of Central Lesion on the Neuronal Number in Ls prepared by dissolving lyophilized NGF in 0.9% phosphate- DRG. The right L5 dorsal roots of newborn rats were tran- buffered saline (Pi/NaCl) at a concentration of 1 mg/ml. The sected on the day of birth (PND-0) and the animals were sac- solution was divided into aliquots and kept frozen at -40C rificed on PND-5. The L5 DRG were dissected out and pre- until use. Subcutaneous injections of either 20 ,ug of NGF or pared for neuronal cell count. The unoperated, contralateral an equal volume of vehicle (P,/NaCl) were given to the neo- DRG served as control. Axotomy of the dorsal roots on nates 2 hr before the operation and continued daily through PND-0 of age caused a 50% decrease of neuronal number by PND-5. NGF and cytochrome c were labeled with 1 mCi of PND-5 (Table 1). There was no decrease of cell number in Na125I (specific activity, 13-17 mCi/pg, Amersham; 1 Ci = the sham-operated animals. 37 GBq) by the lactoperoxidase method of Marchalonis (20) Effects of Combined Central and Peripheral Lesions on the with minor modifications.