Charles P. Barrett

Astroglial reaction in the gray matter of lumbar segments after midthoracic transection of the adult rat spinal cord

Previous studies of cordotomized rats revealed a glial reaction in the gray matter of the spinal cord at sites remote from the lesion, and the present study was done to explore this phenomenon further. Seventy-five young adult female rats were cordotomized and 10 hemicordotomized, both operations at T5. Between 1 and 28 days postoperatively, histologic sections of thoracic and lumbar segments stained by phosphotungstic acid hematoxylin (PTAH, pH 2.37), by periodic acid Schiffdimedon (PAS-D) or by an immunocytochemical method for glial fibrillar acidic protein (GFAP) revealed histological changes as follows: PTAH staining showed that astroglia in thoracic and lumbar regions of the cordotomized rats possessed a swollen, pink-staining cytoplasm and enlarged, thick, dark blue-staining fibrous processes. This response, first noted within 4 days, had intensified by 7 days and was maximal at 14 to 17 days postoperatively. By 28 days, the reaction had diminished but was still readily detectable. The more specific GFAP staining procedure confirmed that the reactive cells were astrocytes and demonstrated that their fibrillar density had increased. The PAS-D reaction revealed glycogen accumulation in glia of the lumbar gray matter within 2 days; this response intensified by 4 days and diminished to normal by 14 days. This reaction was largely concentrated in the perivascular end feet of astroglia, but also appeared in conjunction with perineuronal astroglia. The site of glial reactivity included both dorsal and ventral horns and was particularly noticed in the gray matter surrounding the central canal. In the hemicordotomized rats, the thoracic and lumbar glia response was much more pronounced ipsilaterally than contralaterally. These results support the interpretation that an astroglial response, involving hypertrophy, fibrillogenesis, and glycogen accumulation, occurs in response to degenerating nerve fibers caudal to sites of spinal cord injury.

Differences between adult and neonatal rats in their astroglial response to spinal injury

Transection of the thoracic spinal cord in adult rats produces an astroglial reaction at the lesion site which spreads gradually to lumbar segments. We compared the spread of gliosis in cordotomized adult and neonatal rats in order to evaluate whether or not maturity of long spinal tracts is a precondition for the genesis of this histopathological reaction. By this experiment, we sought to determine whether spread of gliosis is induced by degeneration of nerve fibers in ascending and descending pathways or results from some more general reaction to injury. The spinal cords of 40 neonatal and 30 young adult rats were transected at T5, and 4 to 60 days later the cervical, thoracic, and lumbar segments were examined immunocytochemically for glial fibrillary acidic protein. In the neonatal rats, there was a moderate gliosis at the lesion site by 7 days; this reaction intensified somewhat during the next 60 days but always remained confined to the site of injury. In contrast, the lesion site of adult rats showed a much more intense gliosis; in those animals the response was maximal by 14 days and was characterized by a gradient of decreasing glial reactivity both rostrally and caudally from the transection site. These results support the hypothesis that the spread of gliosis from spinal lesions results from degeneration of the long ascending and descending fiber tracts.

Essentiality of a specific cellular terrain for growth of axons into a spinal cord lesion

To date, there are no reports of growth of significant numbers of axons into or across a lesion of the mammalian spinal cord. However, recent studies showing that CNS axons will grow into PNS environments indicate that comparable growth into spinal cord lesions could be achieved if ischemic necrosis could be prevented and the lesion site repopulated by astrocytes and ependymal cells rather than by the macrophages, lymphocytes, and fibroblasts that generally accumulate at sites of CNS injury. To examine this possibility, we made a laminectomy at T5 in rats and crushed the spinal cord for 2 s with a smooth forceps (leaving the dura mater intact to prevent ingrowth of connective tissue). At 1 week, the lesion was filled with mononuclear cells, degenerating nerve fibers, and capillaries that were oriented parallel to the long axis of the spinal cord. By 2 weeks, longitudinally oriented cords of ependymal cells and astrocytes had migrated into the lesion from the adjacent spinal cord, and similarly oriented nerve fibers had begun to grow into the lesion along these capillaries and cellular cordons. The mononuclear cells had now assumed phagocytic activity and were engorged with myelin and other cellular debris. After 3 weeks, the astrocytes had elaborated thick cell processes. The nerve fibers in the lesion were still oriented longitudinally but had increased in number and were often arranged in small fascicles. These observations provide the first histological evidence of growth of nerve fibers into a lesion of the rat spinal cord. We conclude that the intrinsic regenerative capacity of the spinal cord can be expressed if ischemic necrosis and collagenous scarring are prevented and the spinal cord parenchyma is first reconstructed by its nonneuronal constituents.

Calcium Influx and Activation of Calpain I Mediate Acute Reactive Gliosis in Injured Spinal Cord

Buffering extracellular pH at the site of a spinal cord crush-injury may stimulate axonal regeneration in rats (1; Guth et al., Exp. Neurol. 88: 44-55, 1985). We demonstrated in cultured astrocytes that acidic pH initiates a rapid increase in immunoreactivity for GFAP (GFAP-IR), a hallmark of reactive gliosis (2; Oh et al., Glia 13: 319-322, 1995). We extended these studies by investigating the effects of certain treatments on reactive gliosis developing in situ in a rat spinal cord injury model. A significant reactive gliosis was observed within 2 days of cord lesion in untreated crush or vehicle-treated, crush control animals as evidenced by increased GFAP-IR and hypertrophy of astrocytes. By contrast, infusion of Pipes buffer (pH 7.4) into the lesion site significantly reduced this increase. The increased GFAP-IR appeared to be linked to Ca2+ influx since infusion of a blocker of L-type calcium channels, nifedipine, reduced the ensuing reactive gliosis significantly. While Ca2+ modulates many signaling pathways within cells, its effect on reactive gliosis appeared to result from an activation of calpain I. Calpain inhibitor I, a selective inhibitor of mu-calpain, also significantly reduced reactive gliosis. However, calpain inhibitor II, a close structural analog which blocks m-calpain, had no salutary effect. We suggest, therefore, that the initial reactive gliosis seen in vivo may result from the activation of a neutral, Ca2+-dependent protease, calpain I, through calcium influx.

Repair of the mammalian spinal cord

Abstract In mammals transection of the spinal cord results in permanent paraplegia, whereas in certain lower vertebrates spinal transection may be followed by structural and functional restitution. It is not altogether clear whether this variation in response to injury represents differences in the regenerative capacity of neurons and/or in the ability of the neuronal environment to sustain axon elongation. In this article, we review studies on the pathology of the injured spinal cord in relation to the inherent growth capability of CNS neurons as well as recent experimental approaches designed to provide an environment conducive to axonal elongation.

Transport of acid phosphatase in normal and transected rat sciatic nerve

Abstract Acid phosphatase accumulates at the severed ends of transected peripheral nerve or spinal cord. We examined the temporal sequence of enzyme accumulation from 1 to 14 days after transecting or applying colchicine to the rat sciatic nerve in order to ascertain whether or not this accumulation is related to axonal transport. Enzyme activity was observed in the proximal and distal stumps by 1 day after transection, and by 1 week activity in the proximal stump exceeded that in the distal stump. In some experiments, colchicine was applied to the nerve proximal to the site of nerve transection. This procedure resulted in an accumulation of acid phosphatase proximal to the site of drug application and not immediately proximal to the site of transection. These results are consistent with the hypothesis that bidirectional transport of acid phosphatase is a normal feature of peripheral nerves.

Enzyme histochemical changes after transection or hemisection of the spinal cord of the rat

Abstract The spinal cords of 84 young adult female rats were transected or hemisected at T7 to T8 and the animals autopsied at intervals from 6 h to 14 months postoperatively. Frozen sections of the unfixed spinal cord on either side of the lesion were prepared for enzyme histochemistry, immunocytochemistry, and histology. The most striking enzymatic alterations and their physiological implications were: (i) (Na+K+)-activated ATPase activity decreased in axons of the gray and white matter within 6 h after spinal transection and did not return subsequently, whereas the decrease in activity that occurred contralateral to a hemisection was transient. The decreased activity occurred so promptly as to suggest possible roles in the genesis of the initial flaccid paralysis (spinal shock) in the spinal animal and in the temporary paraplegia seen after subtotal spinal injury. (ii) During the first week postoperatively, many axons in the white matter developed large swellings or small varicosities that reacted strongly only for enzymes normally present in the neuronal perikaryon (e.g., AChE, acid phosphatase, NADH-diaphorase, and G6PDH). This histopathological reaction gradually spread rostrally and caudally from the site of injury, but it disappeared as axonal degeneration supervened. (iii) Within 7 days after spinal transection, many neuronal perikarya were chromatolytic and exhibited decreased AChE activity but normal or increased NADH-diaphorase activity. This response is similar to that seen in the cell bodies of regenerating peripheral axons where anabolic processes are favored over neurotransmission-related functions. (iv) Increased cellularity of the spinal parenchyma adjacent to the lesion resulted largely from the proliferation and hypertrophy of astrocytes. These hypertrophied cells, whose identity was confirmed by GFAP immunocytochemistry, reacted with marked intensity for NADH-diaphorase, G6PDH, and Gly3PDH. Such enzyme changes, characteristic of increased protein turnover, indicate that experimental attempts to control gliosis (e.g., by reducing protein turnover or by other means) could be effectively monitored by enzyme histochemistry.

The mechanism by which degenerating peripheral nerve produces extrajunctional acetylcholine sensitivity in mammalian skeletal muscle

Abstract Previous studies by others showed that a graft of peripheral nerve, placed on the surface of a muscle, induced acetylcholine hypersensitivity in the underlying muscle fibers, and it was therefore suggested that denervation-induced acetylcholine sensitivity also results from the release of substances from the degenerating peripheral nerve rather than from the loss of neurotrophic influences. To examine the basis for this hypothesis, we repeated the experiments, and in addition to the biophysical investigation, we also included histological analysis of the muscle. An autograft of brachial nerve was placed on the soleus or extensor digitorum longus muscle of adult rats. Three days postoperatively, the muscle fibers underlying the graft and immediately adjacent to that site were depolarized by 30% and exhibited extrajunctional acetylcholine sensitivity. Histopathological changes accompanied these physiological abnormalities. Within 2 days postoperatively an inflammatory response was seen and vacuoles appeared in the sarcoplasm. By 3 days the vacuoles had enlarged and coalesced so that many muscle fibers appeared devoid of sarcoplasm. By 4 days, regeneration was initiated by the fusion of mononuclear cells which gave rise to syncytial cells, and by 7 days these elements had developed into typical basophilic myotubes with central nuclei. Although certain diseases such as muscular dystrophy display a histopathological picture of degeneration and regeneration, this is not found after denervation (which is characterized by atrophy rather than degeneration of muscle fibers). The present findings therefore do not support the hypothesis that products of degenerating nerve play a role in the genesis of denervation hypersensitivity and depolarization.

Formation of Virus-like Particles by Bone Cells in Mice with a High Incidence of Spontaneous Leukemia

Bone samples from potentially leukemic and leukemic mice revealed numerous 90-to 110-nanometer particles morphologically identical to murine leukemia virus. Particles were observed budding from plasma membranes of osteocytes and osteoblasts but were most numerous in osteocyte lacunae. Particles were not observed in bone samples from mice which rarely develop leukemia.

A possible non-oncogenic effect of C type bone cell virus on serum calcium levels of potentially leukemic mice

In bone of C3H/Fg mice, particles structurally identical toC-type leukemia virus arise from membranes of osteocytes and osteoblasts. Although these virus apparently do not induce morphologic or neoplastic change in bone they may have other, more subtle, effects. Thus, comparison of sera form male C3H/Fg mice, a high leukemia-prone strain, with C57BL and C3H/HeJ mice, low leukemia strains which do not containC-type virus in bone, reveals that serum calcium levels are significantly lower in the former than in the latter. Further, when C3H/Fg mice develop frank leukemia there is a corresponding increase in virus particles while the serum calcium concentration levels fall to even lower values. The presence of leukemia itself appears not to be the cause as indicated by the failure of implanted lymphocytic leukemic cells in C3H/Fg mice to significantly affect serum calcium concentration. It is postulated that the effects of the virus could be due either to increased osteoblastic activity or to inhibition of osteocytic osteolytic activity or to both.