Restoration of conduction and growth of axons through injured spinal cord of neonatal opossum in culture.

Abstract

The ability of neurons in the central nervous system to grow through a lesion and restore conduction has been analyzed in a developing spinal cord. The preparation consists of the entire central nervous system of the newly born opossum (Monodeiphis domestica), isolated and maintained in culture. Cell division, cell migration, and reflexes are maintained in such preparations for up to 8 days in culture. In the present experiments, massive lesions were produced by crushing the spinal cord, which abolished all conduction for a day. By 2-3 days after injury, electrical conduction across the crush could be observed. After 4-5 days, clear recovery had occurred: the amplitude of the conducted volley was comparable to that in acute preparations. In such preparations, the spinal cord had largely regained its normal appearance at the crush site. Axons stained by carbocyanine dyes or horseradish peroxidase had, by 4 days, grown in profusion through the lesion and several millimeters beyond it. These experiments demonstrate that neurons in the central nervous system of newly born mammals, unlike those in adults, can respond to injury by rapid and extensive outgrowth in the absence of peripheral nerve bridges or antibodies that neutralize inhibitory factors of myelin. With rapid and reliable regeneration occurring in vitro, it becomes practicable to assay the effects of molecules that promote or inhibit the restoration of functional connections.Damage to the central nervous system (CNS) of adult mammals is usually followed by minimal regrowth or repair (1). Axons can regenerate and make connections with their targets only after grafts of peripheral nerves have been supplied (2-4) or after inhibitory growth molecules have been neutralized (5-7). Oligodendrocytes in particular have been shown to produce molecules that actively prevent neurons from growing. By contrast, neurons within the CNS of a developing mammalian embryo are unmyelinated, grow profusely, and form synapses in the virtual absence of glial cells. The aim of the present experiments was to determine whether damaged immature mammalian CNS might be better able to repair itself after an injury. Such tests have not been made on embryos in the past, for technical reasons. Although axonal outgrowth can occur in neonatal hamsters following injury to pyramidal-tract fibers, the regenerating axons are not able to grow across the lesion (8).A suitable experimental preparation for studying regeneration after an injury is provided by the central nervous system of the neonatal opossum (Monodelphis domestica). This animal, being a marsupial, is extremely immature at birth. The newly born pup has only rudimentary eyes, ears, and hindlimbs and is unable to walk or to right itself. Its CNS is incomplete and corresponds to that of a 14-day rat embryo, having no cerebellum and only a rudimentary forebrain (9). Yet this neonatal animal is able to suck, to breathe, and to perform selected vital functions. Earlier experiments (10-12) have shown that the CNS can be removed in ...

Electroanatomic Mapping of the Intercaval Bundle in Atrial Fibrillation

“…Although not seen in humans, this has been reported in the possum and zebrafish. 10,11 Of note, standard 30 day transtelephonic monitors worn 3 months after the ablation procedures revealed no recurrence of atrial arrhythmias in all 4 patients.…”

Myelin-associated neurite growth-inhibitory proteins and suppression of regeneration of immature mammalian spinal cord in culture.

“…In DRG preparations the culture medium contained NGF at 120 ng/ml. The crushes caused complete disruption of all neurites on that side of the spinal cord (26,27 (5), these tracts become myelinated late and regeneration continues until day 16 (5,33).…”

“…Opossum (Monodelphis domestica) pups were taken from the colony at 6, 13, 15, and 17 days after birth, anesthetized by methoxyflurane and cooling on ice, and killed by rapid section of heart and lungs (5,(24)(25)(26)(27). The entire CNS was removed and cultured in basal medium Eagle (BME; GIBCO) gassed with 95% 02/5% CO2.…”

“…The CNS in its entirety survives well for periods of a week or longer and continues to exhibit reflex behavior, cell division, and growth in culture (24,25). Moreover, repair that is rapid (5 days), extensive, and reliable occurs after a crush or a cut has been made to the spinal cord in vitro, provided the animal is less than 12 days of age (26)(27)(28). In this preparation it has been shown that the capacity for regeneration across a cervical spinal cord lesion comes to an abrupt end at 12 days, the stage at which astrocytes, oligodendrocytes, myelin and myelin-associated growth-inhibitory proteins have developed (5).…”

Regeneration in the central nervous system

“…Interestingly, successful regeneration giving rise to functional recovery is seen in the CNS of young mammals (Bates and Stelzner, 1993;Hasan et al, 1993). Defined spinal cord lesions in chicken embryos or newborn hamsters, opossums, cats or rats lead to extensive growth of regenerating fibers into the denervated spinal cord, whereas no such growth is observed after lesions in the adult (Kalil and Reh, 1982;Tolbert and Der, 1987;Hasan et al, 1991;Treherne et al, 1992;Bregman et al, 1993;Saunders et al, 1998). Thus, adult neurons seem to loose this ability during late postnatal development and the regenerative response becomes abortive.…”