Barbara S. Bregman

Development of serotonin immunoreactivity in the rat spinal cord and its plasticity after neonatal spinal cord lesions

The postnatal maturation of spinal pathways may account for the gradual time course of postnatal development of behavior and also account for the greater anatomical reorganization which often follows damage to the developing CNS compared to the mature CNS. The purpose of the current study was to examine (1) the prenatal and postnatal development of the descending serotonergic (5-HT) projection to the spinal cord and (2) the effects of a neonatal spinal cord lesion on this development. In addition, we wished to determine (3) whether transplants of fetal spinal cord tissue placed into the neonatal lesion site alter the plasticity of the 5-HT projection to the cord. Peroxidase-antiperoxidase immunocytochemical techniques were used. At embryonic day 14 (E14), no 5-HT immunoreactive fibers could be identified at any spinal cord level. By E18 the first axons were identified in the white matter only at all spinal cord levels. At birth, 5-HT immunoreactive fibers were present both in the white matter and in the gray matter at all cord levels. The projection within the gray matter was diffuse and considerably less dense than in the adult. The postnatal maturation of the 5-HT projection within the gray matter of the spinal cord followed rostral to caudal and ventral to dorsal gradients. During the first weeks postnatal, the 5-HT immunoreactivity within the cord increased to attain an adult pattern and density by 14 days in the cervical cord and 21 days in the thoracic and lumbar cord. The effect of a spinal cord hemisection at birth on the anatomical reorganization of the descending serotonergic innervation of the cord was compared with the effect of the same lesion in the adult. In the adult animal, mid-thoracic hemisection decreased the 5-HT content of the ventral horn of the lumbar spinal cord caudal and ipsilateral to the lesion to 8% of that on the intact side. When this same lesion was made in the newborn animal, the innervation was 43% of that on the intact side. When a transplant of fetal spinal cord tissue was inserted into the lesion site in the newborn animals, there was even greater 5-HT innervation caudal to the lesion, 83% of that on the intact side. These results indicate that there is considerable postnatal development and plasticity of the descending serotonergic projection to the spinal cord, and this plasticity is enhanced by the presence of a spinal cord transplant at the site of the lesion.

Spinal cord transplants permit the growth of serotonergic axons across the site of neonatal spinal cord transection

These experiments were designed to determine whether transplants of fetal spinal cord tissue into lesioned spinal cord in newborn rats provide a terrain that supports the growth of serotonergic (5-HT) axons across the site of the lesion. Although descending serotonergic axons can regenerate after chemical lesions in adult animals, they show little regrowth after surgical lesions. In newborn animals, 5-HT axons do not regrow after either chemical or mechanical lesions since the axotomized raphe-spinal neurons die. After partial spinal cord lesions made in developing animals, immature axons can take an aberrant route around the site of the lesion to reach normal target areas. Even these robust, late-growing, uninjured axons, however, are unable to grow through the site of the spinal cord lesion. Immunocytochemical labeling was used to determine if descending serotonergic axons grow into fetal spinal cord transplants, and whether these axons cross the transplant to reach spinal cord levels caudal to the lesion. Spinal cord transection at a mid-thoracic spinal cord level on the day of birth resulted in a dramatic decrease in 5-HT immunoreactivity caudal to the lesion by one day postoperative. 5-HT immunoreactivity caudal to the lesion was abolished by 5 days postoperative and did not return after acute or chronic (6 months) survival periods. When a transplant of fetal spinal cord tissue was placed into the lesion site, 5-HT axons were identified throughout the transplant. At spinal cord levels caudal to the transection and transplant, the serotonergic axons were identified in the host spinal cord in both the white and gray matter. This 5-HT innervation was not confined to spinal cord segments adjacent to the lesion site but extended to spinal cord segments as far as lower lumbar levels. The reinnervation of the host spinal cord caudal to the transection was far less than that seen in unlesioned adult rat spinal cord. Horseradish peroxidase (HRP) injected caudal to the transection and transplant, retrogradely labeled neurons within the medullary raphe nuclei. The HRP and 5-HT results both depended on apposition of the transplant with the rostral and caudal stumps of the host spinal cord; without such apposition, labeling was abolished. These results indicate that the presence of a transplant at the site of the neonatal lesion modifies the environment at the lesion site in such a manner as to support the elongation of identified axons across the site of the lesion and into the host cord caudal to the lesion.

Transplants Enhance Locomotion in Neonatal Kittens Whose Spinal Cords Are Transected: A Behavioral and Anatomical Study

We have studied the locomotor development of kittens that received complete low thoracic spinal cord transections and embryonic spinal cord transplants as newborns. Embryonic spinal cord (E21-E26) transplanted into the site of a transection integrated well with the host spinal cord and promoted the development of overground locomotion. Spinalized kittens with transplants were first distinguished from spinalized kittens during the 2nd and 3rd postnatal weeks when kittens with transplants positioned their hindlimbs underneath their bodies which promoted support of the hindquarters. By postnatal Week 6, kittens with transplants exhibited overground locomotion characterized by full weight support and moderate balance control. By 20 weeks of age, as many as 96% of the step cycles showed full weight support and as few as 2% of the step cycles were interrupted by a fall. Most kittens also showed coordination between the forelimbs and the hindlimbs. They differed from normal in the precocious onset of reflex stepping and in the less precise interlimb coordination and more precarious balance during overground locomotion. The overground locomotor performance of kittens with transplants greatly exceeded that of spinal kittens without transplants since few spinalized kittens showed any full-weight-supported step cycles and none showed coordination between the forelimbs and the hindlimbs. In the absence of a transplant, no fibers could grow across the lesion site. In the presence of a transplant, fibers grew across the lesion site and established anatomical connectivity with the host. Host segmental systems identified by the presence of calcitonin gene-related peptide- and substance P-immunoreactive fibers were found throughout the transplants. Descending host systems of supraspinal origin were identified by serotonin- and dopamine beta-hydroxylase-immunoreactive fibers throughout the transplants. The growth of supraspinal axons into the transplant, and in one case into the caudal host spinal cord, provided a possible anatomical basis for the development of coordinated overground locomotion.

CNS transplants promote anatomical plasticity and recovery of function after spinal cord injury

We are using neural tissue transplantation after spinal cord injury to identify the rules which determine the response of young neurons to injury, to identify the mechanisms underlying anatomical plasticity and recovery of function following spinal cord injury, and to determine the conditions which change during development, leading to the more restricted growth capacity of mature neurons following injury. Spinal cord lesions at birth interrupt different pathways at different relative stages in their development. Neural tissue transplants modify the response of the immature central nervous system neurons to injury. In the current studies, we have used neuroanatomical and behavioral methods to compare the response of the late-developing corticospinal pathway with that of brainstem-spinal pathways which are intermediate in their development and that of the relatively mature dorsal root pathway. We find that both late-developing and regenerating neuronal populations contribute to the transplant-induced anatomical plasticity, and suggest that this anatomical plasticity underlies the transplant-mediated sparing and recovery of function.

Differentiation of substantia gelatinosa-like regions in intraspinal and intracerebral transplants of embryonic spinal cord tissue in the rat

The differentiation of intracerebral and intraspinal transplants of fetal (E14-E15) rat spinal cord was studied to determine the extent to which myelin-free zones in these embryonic grafts exhibit cytological features and immunocytochemical characteristics of the substantia gelatinosa (SG) of the normal spinal cord. Immunocytochemical staining with antiserum to myelin basic protein (MBP) revealed myelin-free areas of varying proportions within fetal spinal cord grafts. These regions were identified in both newborn and adult recipients regardless of whether donor tissue was grafted to heterotopic (intracerebral) or homotopic (intraspinal) sites. As in the SG of the intact spinal cord, the myelin-free regions consisted mainly of small (7-15 microns) diameter neurons. At the ultrastructural level, these cells were surrounded by a neuropil composed of numerous small caliber, unmyelinated axons and intermediate-sized dendrites. Synaptic terminals in these areas were primarily characterized by the presence of clear, round vesicles, although granular vesicles were occasionally found within these terminals. Immunocytochemical staining demonstrated met- and leu-enkephalin-, neurotensin-, substance P-, and somatostatin-like immunoreactive elements within these myelin-free areas. Thus, regions within embryonic spinal cord grafts undergo some topographical differentiation which parallels that of the normal superficial dorsal horn. The presence of SG-like regions illustrates the potential capacity of fetal spinal cord transplants for replacing some intraspinal neuronal populations at the site of a spinal cord injury in neonatal and adult animals. These graft regions may serve as a source of intersegmental projection neurons or establish an extensive intrinsic circuitry similar to that seen in the normal SG. In addition, the definition of these areas provides a useful model to study the innervation patterns of host axons that typically project to the substantia gelatinosa of the normal spinal cord.

Development of locomotor behavior in the spinal kitten

This study was undertaken to determine the locomotor capability of kittens whose spinal cords were transected at birth. The postnatal development of reflex and goal-directed locomotion was examined during the first 5 postnatal months in kittens that received low thoracic spinal cord transections as newborns. Some spinal kittens developed aberrant quadrupedal forms of locomotion. The onset of quadrupedal locomotion, however, was delayed by 2-3 months compared to the normal kitten (42) and deteriorated by 5 months of age. Qualitative and quantitative analyses demonstrated that the quadrupedal locomotion was abnormal. Although some step cycles were characterized by full weight support, the typical hindlimb step cycle of the best performing cat showed inadequate weight support and balance. No spinal cat was able to coordinate the hindlimbs with the forelimbs during overground locomotion on a runaway or during quadrupedal locomotion on a treadmill. Neuroanatomical tracing with WGA-HRP and immunocytochemical techniques showed no axonal regeneration or growth into or across the lesion sites. The aberrant form of quadrupedal locomotion developed without descending input to the caudal spinal cord. The variability in performance among animals suggested that compensatory strategies were important factors in the spinal kittens achievement of quadrupedal locomotion. Hindlimb weight-supported stepping during quadrupedal locomotion in some animals underscored the capacity of the isolated caudal spinal cord to generate both rhythmical stepping movements and weight support. The maintenance of developmentally immature, but functional, hindlimb postures suggested that the development of the isolated caudal spinal cord was arrested in the absence of descending input.

Chapter 26 Effect of target and non-target transplants on neuronal survival and axonal elongation after injury to the developing spinal cord

Publisher Summary This chapter determines: (1) whether transplants of fetal spinal cord tissue at the lesion site can prolong the critical period for developmental plasticity of the corticospinal system, and (2) whether the requirements for survival of immature axotomized neurons are target specific, or whether a variety of immature tissues can substitute for the normal target and support these neurons after injury. Target-specific transplants extend the critical period for developmental plasticity of the corticospinal pathway beyond that seen after lesions alone are demonstrated in the chapter. The corticospinal (CS) axons damaged prior to synaptogenesis are found to exhibit a greater degree of growth than those injured after synaptogenesis is completed, suggesting that an interaction of environmental and neuronal factors regulate the capacity of immature corticospinal neurons for growth. Many immature CNS neurons undergo massive retrograde cell loss in response to target removal by axotomy. At acute survival times, both target and non-target transplants are able to support the temporary survival of axotomized red nucleus (RN) neurons, perhaps by providing a diffusable trophic support for the injured neurons. However, their permanent survival is supported only by target-specific transplants, and they may require axonal elongation and synaptogenesis.

Delayed rehabilitation with task-specific therapies improves forelimb function after a cervical spinal cord injury

PURPOSE The effect of activity based therapies on restoring forelimb function in rats was evaluated when initiated one month after a cervical spinal cord injury. METHODS Adult rats received a unilateral over-hemisection of the spinal cord at C4/5, which interrupts the right side of the spinal cord and the dorsal columns bilaterally, resulting in severe impairments in forelimb function with greater impairment on the right side. One month after injury rats were housed in enriched housing and received daily training in reaching, gridwalk, and CatWalk. A subset of rats received rolipram for 10 days to promote axonal plasticity. Rats were tested weekly for six weeks for reaching, elevated gridwalk, CatWalk, and forelimb use during vertical exploration. RESULTS Rats exposed to enriched housing and daily training significantly increased the number of left reaches and pellets grasped and eaten, reduced the number of right forelimb errors on the gridwalk, increased right forelimb use during vertical exploration, recovered more normal step cycles, and reduced their hindlimb base of support on the CatWalk compared to rats in standard cages without daily training. CONCLUSIONS Delayed rehabilitation with enriched housing and daily forelimb training significantly improved skilled, sensorimotor, and automatic forelimb function together after cervical spinal cord injury.

Recovery of Locomotion and Skilled Forelimb Function After Spinal Cord Injury in Rats: Effects of Transplants and Neurotrophic Factors

Spinal cord injury (SCI) removes descending input to spinal cord circuitry and results in loss of function below the level of injury. Interventions designed to increase the intrinsic capacity of CNS neurons for growth and other interventions to create a favorable environment at and below the lesion site result in increased regrowth and recovery of function. It is unlikely that any single intervention will restore function completely. After SCI in rats, transplants of fetal spinal cord tissue and the exogenous application of neurotrophic factors restore supraspinal input and permit recovery of skilled movement and locomotion. Delay of treatment leads to greater axon regeneration and recovery of function than immediate application of transplants and neurotrophic factors.

Neural Tissue Transplants Modify Response of the Immature Spinal Cord to Damage

Neural tissue transplantation techniques have been used extensively in recent years to examine questions concerning development and regeneration in the developing and mature central nervous system (see 1,2,3, for reviews). For example, transplants derived from various levels of the neuraxis survive, grow, and differentiate when placed into cavities or lesions in the adult or newborn nervous system (4–13). Often anatomical projections between host and transplant tissue are established. In some instances, the transplants are able to mediate recovery of function after CNS lesions either by establishing axonal connections with the host nervous system or by releasing hormones or neurotransmitters which are able to act on neurons within the host (3, 7, 14-19).