Dennis J. Stelzner

Effects of spinal transection in neonatal and weanling rats: Survival of function

Abstract The spinal cord of neonatal rats and weanling rats was transected midthoracically. The recovery and maturation of responses in the hind quarters were followed and compared with response development in normal rats. In rats transected as neonates, the hind limbs supported the hind quarters, the tail was dorsiflexed, and the hind limbs stepped during locomotion. When a hind limb fell through a grid, struggling movements, replacement, and a response similar to tactile placing were observed. Rapid extension often bounced the hind quarters off a grid when the foot pads contacted a grid bar. Pinching elicited clonic flexion and wriggling of the hind quarters with a prolonged afterdischarge. In animals transected as weanlings, the hind limbs and tail were passively extended and did not participate in locomotion. The hind limbs also hung passively through a grid and struggling was brief. Clonic flexion and wriggling to pinch lacked an afterdischarge. Choreoathetoidlike movements and spasms were common in the weanling group. In a number of respects including temporal correspondence in the appearance of certain responses, the response maturation of the neonatally transected animal resembled the ontogeny of responses in the normal animal. These data indicate that the local development of the spinal cord continues in the absence of supraspinal control. Hence it appears that the presence or absence of supraspinal control during development is the basis for the differences in the survival of responses from the isolated spinal cords of the neonatal group when compared to the weanling animals. This “survival of function” effect may help to explain the sparing of function often found after lesions in the developing nervous system.

Laminin distribution during corticospinal tract development and after spinal cord injury

The glycoprotein laminin is a prominent constituent of basal laminae and has been suggested to play an important role in axonal growth. We have tested this hypothesis, by examining the temporal and spatial distribution of laminin in the rat spinal cord, relative to elongating corticospinal tract (CST) axons, during normal development and after newborn and adult spinal lesions. The distribution of laminin was demonstrated in spinal cord sections from animals ranging in age from 14 days embryonic to adult using immunocytochemistry. Anti-laminin immunolabeling was seen around blood vessels and meninges in all the animals examined. However, within the grey and white matter its distribution was age-dependent. In the normal cord, immunostaining appeared in small amounts in early embryos, but was absent from all postnatal animals even at ages when the CST was growing down the cord. Following injury, intense immunostaining was associated with lesions in both newborn and adult operates at all postoperative periods examined. Within the matrix of the lesion laminin immunostaining was especially prominent. In the intact cord it was prominent only around blood vessels near the lesion site. Our results indicate that the distribution of laminin does not closely correlate with axonal growth of the CST either during normal development or after spinal injury.

Do propriospinal projections contribute to hindlimb recovery when all long tracts are cut in neonatal or weanling rats

Lateral hemisection lesions separated by 1 to 3 spinal segments were made on opposite sides of the mithoracic spinal cord in 1-month-old (N = 15; weanling operates) and newborn albino rats (N = 16; neonatal operates). Hindlimb behavior was assessed between 1 and 6 months p.o. for both groups of operates using a protocol and rating system that have previously proved effective in differentiating behavioral recovery of the hindlimbs as a function of age of spinal transection. In addition, at the conclusion of behavioral testing, operates received spinal injections of [3H]proline and HRP caudal to the spinal lesions to determine if lesions were complete and if neurons within the region between the two lesions (interlesion zone) projected into the caudal spinal cord. In both groups of operates, neurons were retrogradely labeled within the interlesion zone bilaterally, primarily in laminae VII-VIII. When both lesions were complete lateral hemisections in weanling operates, little behavioral recovery was observed, similar to complete spinal cord transection (N = 3). However, much greater behavioral recovery was seen, including supporting reactions and locomotor responses, when one or both lesions spared axons along the ventrolateral rim of the white matter. Neurons were retrogradely labeled in the brain stem reticular formation (N = 12) in these cases. All lesions were complete lateral hemisections in neonatal operates but much greater behavioral recovery was seen than in weanling operates with the same lesions, including supporting, placing, and locomotor responses. In an additional group of eight neonatal operates, the spinal cord rostral to the spinal hemisections was transected at 1 month of age. Supportive, placing, and locomotor responses were seen immediately after recovery from anesthesia and responses returned to pretransection levels in six of eight operates over the 10-day survival period. Fink-Heimer impregnation showed that degeneration argyrophilia from the transection bilaterally filled the interlesion zone but little argyrophilia was seen caudal to this region. Our results indicate that an intact propriospinal circuit remains in both neonatal and weanling operates but does not appear to contribute to hindlimb response development or recovery. The greater behavioral recovery in neonatal operates appears due to intrinsic connections (doral root, interneuronal) continuing to be able to drive the spinal circuitry underlying the spared behaviors.

Lack of intralaminar sprouting of retinal axons in monkey LGN

In the dorsal lateral geniculate nucleus (LGN) of the adult cat there is no evidence for translaminar sprouting of retinal axons to fill sites freed of retinal endings from the other eye. We tested the possibility that retinal axons will sprout to fill denervated retinal sites within laminae of the monkey LGN. In 4 monkeys, retinal ganglion cell axons from either the upper or lower half of the retina were destroyed. To maximize the potential for sprouting in the LGN, on one side of the brain the LGN cells to which the remaining retinal axons normally project were removed by ablation of the appropriate portion of the striate cortex. Three months later the eye receiving the retinal lesion was injected with [3H]proline and the retinal projection to the LGN on both sides of the brain was studied using autoradiography. We found no evidence of intralaminar sprouting of retinal axons either in the normal LGN or in the LGN in which the usual targets of retinal axons had been removed.

NMDA antagonism during development extends sparing of hindlimb function to older spinally transected rats

Hindlimb weight support and bipedal stepping occur after spinal cord transection in neonatal rats (birth to 12 days of age) while the same lesion in 15-day and older animals results in permanent loss of these responses. Some compensatory change in lumbar spinal circuitry must occur after spinal transection in young animals subserving these hindlimb behaviors. In contrast, animals just a few days older are incapable of such compensatory responses. We have examined the hypothesis that neural activity leads to the postnatal loss of plasticity in spinal circuitry. We find that antagonism of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor with MK-801 in young animals extends the sparing of hindlimb function after spinal transection to older animals. This effect is not due to a non-specific depression of all exciatory drive to motor neurons since Ia to motor neurons synaptic transmission through non-NMDA receptors is preserved during MK-801 treatment. Acute administration of MK-801 at the time of spinal transection or chronic administration of MK-801 after postnatal day 17 has no effect on recovery of hindlimb function after spinal transection. These results highlight the importance of NMDA receptor activation in spinal circuit maturation.

Attempts to facilitate dorsal column axonal regeneration in a neonatal spinal environment.

The response to injury of ascending collaterals of dorsal root axons within the dorsal column (DC) was studied after neonatal spinal overhemisection (OH) made at different levels of the spinal cord. The transganglionic tracer, cholera toxin conjugated to horseradish peroxidase, and the anterograde tracer, biotinylated dextran amine, were used to label dorsal root ganglion cells with peripheral axons contributing to the sciatic nerve. There was no indication of a regenerative attempt by DC axons at acute survival times (3 days and later) after cervical injury, replicating previous work done at chronic survival periods (Lahr and Stelzner [1990] J. Comp. Neurol. 293:377–398). There was also no evidence of DC regeneration after lumbar OH injury even though immunohistochemical studies using the oligodendrocyte markers Rip and myelin basic protein showed few oligodendrocytes in the gracile fasciculus at lumbar levels at birth. Therefore, the lack of myelin in the dorsal funiculus at lumbar levels does not enhance the growth of neonatally axotomized DC axons. In addition, DC axons did not regenerate when presented with fetal spinal tissue implanted into thoracic OH lesions, even though positive control experiments showed that segmental dorsal root axons containing calcition gene‐related peptide and corticospinal axons grew into these implants, replicating previous work of others. When a thoracic OH lesion, with or without a fetal spinal implant, was combined with sciatic nerve injury to attempt to stimulate an intracellular regenerative response of DRG neurons, again, no evidence of DC axonal regeneration was detected. Quantitative studies of the L4 and L5 dorsal root ganglia (DRG) showed that OH injury did not result in DRG neuronal loss. However, sciatic nerve injury did result in significant post‐axotomy retrograde cell loss of DRG neurons, even in groups receiving thoracic embryonic spinal implants, and is one explanation for the minimal effect of sciatic nerve injury on DC regeneration. Although fetal tissue did not appear to rescue a significant number of DRG neurons, the quantitative analysis showed an enlargement of the largest class of DRG neuron, the class that contributes to the DC projection, in all groups receiving fetal tissue implants. This apparent trophic effect did not affect DC regeneration or neuronal survival after peripheral axotomy. Further studies are needed to determine why DC axons do not regenerate in a neonatal spinal environment or within fetal tissue implants, especially because previous work by others in both the developing and adult spinal cord shows that dorsal root axons will grow within the same type of fetal spinal implant.

Regeneration of the Frog Optic Nerve Comparisons With Development

Developing and regenerating frog optic axons grow within optic pathways and form connections only with optic targets. However, unlike normal development, many regenerating optic axons in the adult frog are misrouted within optic pathways, including axons that grow into the opposite retina. Many of the axons misrouted during regeneration appear to be collaterals of axons that grow in normal directions. Ganglion cell loss of up to 60% occurs after optic nerve damage, beginning prior to reinnervation of optic targets. Massive axonal collateralization also takes place near the point of nerve damage, causing the normal order found within the nerve to be lost. Collaterals are eliminated as selective reinnervation is completed, and the smaller complement of optic cell axons remaining after regeneration form an expanded projection within optic targets. Evidence is reviewed that suggests that factors involved in axonal guidance and target recognition during development remain intact in the adult frog brain. Additional conditions resulting from nerve injury causes axonal guidance to be less successful during regeneration.

Increase in ganglion cell size after optic nerve regeneration in the frog, Rana pipiens

Even though optic regeneration is successful in the frog, Rana pipiens, at completion considerable ganglion cell loss has occurred. To determine whether ganglion cell loss affects the size of the remaining ganglion cells, these cells were back-filled with horseradish peroxidase. The size of one class of ganglion cell 6 months to 1 year following nerve crush injury (N = 4) was compared to that of normal cells of this class (N = 4). The average area of the perikaryon was 35% larger than normal (less than 0.01). This change is interpreted to reflect the increased metabolic needs of the neuron required to maintain a larger than normal axonal arbor.

Frog tectal efferent axons fail to regenerate within the CNS but grow within peripheral nerve implants.

Tectal efferent axons, located adjacent to the optic tract, fail to regenerate past diencephalic lesions in Rana pipiens even though optic axons regenerate after the same injury (M. J. Lyon and D. J. Stelzner, J. Comp. Neurol. 255: 511-525). We tested the possibility that tectal efferent axons can regenerate within peripheral nerve implants. A 6- to 8-mm segment of autologous sciatic nerve was implanted into the anterolateral (N = 23) or centrolateral (N = 22) portion of the dorsal surface of the tectum. Frogs survived for 6 (N = 16) or 12 weeks (N = 29) before the free end of the nerve was recut and HRP applied. A control group had the nerve crushed prior to the HRP application. Neurons within the tectum, near and medial to the implant site, were retrogradely labeled from the nerve graft in most experimental operates but no neurons were labeled in controls. In addition, neurons were also labeled in nuclei which projected to the tectum in a number of cases. Three times as many neurons were labeled in 12-week operates (42 +/- 46) as in 6-week operates (15 +/- 12). The morphology and location of labeled neurons in the tectum was similar to tectal efferent neurons except that the somal area of neurons labeled from the graft was significantly larger (41%) than normal tectal efferent neurons. The basic finding is similar to experiments using the same paradigm in the mammalian central nervous system (CNS). One difference is the minimal glial reaction at the graft insertion site.(ABSTRACT TRUNCATED AT 250 WORDS)