Edward J. Donati

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.

Carbonic anhydrase activity in first-order sensory neurons of the rat.

The localization of carbonic anhydrase within nervous tissues is to some extent controversial and, despite the general agreement that enzyme activity is present in choroid plexus and neuroglia and absent from neuronal perikarya, it is still uncertain whether all glial cells (astrocytes, oligodendrocytes, satellite cells, and Schwann cells) are reactive and all neurons nonreactive. We took advantage of a recently described improvement of the light microscopic method to reexamine this subject and, in the course of this study, observed an unusually selective neuronal localization of enzymatic activity: some of the large and medium-sized neurons of the rat’s spinal, nodose, and trigeminal ganglia reacted positively for carbonic anhydrase, whereas other peripheral and central neurons did not. The perikarya and the central and peripheral processes of the reactive cells were clearly visualized by the histochemical reaction. The central processes were easily traced from their origin in the spinal ganglion into the dorsal columns of the spinal cord and to their terminations in the gray matter of the spinal cord and the gracile and cuneate nuclei of the med-

Origin of the connective tissue scar in the transected rat spinal cord

Abstract The dense collagenous tissue that invades the site of spinal transection or hemisection in the rat constitutes a physical impediment to axonal regeneration and prevents functional reunion of the cut ends of the spinal cord. If this connective tissue response in the injured spinal cord were prevented, one barrier to regeneration would be eliminated, and we could better evaluate the role of other factors which affect regeneration of spinal axons. In the present study, the transected and hemisected spinal cords of rats were covered on the dorsal surface with a synthetic dural sheath composed of dacron and silicone. The dural sheath significantly reduced the invasion of connective tissue into the site of transection or hemisection. Because the sheath was located extradurally, we conclude that the fibroblasts which give rise to the connective tissue scar derive not from the dura mater but from connective tissue components of injured bone and muscle tissue adjacent to the spinal cord.

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.

Induction of intramuscular collateral nerve sprouting by neurally applied colchicine

Abstract The plantaris muscle of the rat is innervated by fibers deriving from spinal nerves L4 and L5. When L4 is transected, the intact residual L5 fibers sprout intramuscular preterminal processes which reinnervate some of the denervated muscle fibers and restore their weight and strength. Experiments by Diamond and his colleagues on cutaneous innervation in salamanders indicated that collateral sprouting can be elicited by applying colchicine to a nerve as well as by transecting it, and it therefore seems that collateral sprouting results from the interruption of axonal transport rather than from nerve degeneration. We tested this hypothesis by either transecting or applying colchicine to spinal nerve L4 in the rat and measuring the isometric strength of contraction of the plantaris 2 weeks later. After transection of L4, electrical stimulation of L4 gave no response whereas stimulation of L5 gave a supranormal isometric tension; after application of colchicine to L4, stimulation of L4 resulted in a normal isometric tension and stimulation of L5 gave a supranormal one. The muscles were examined histologically by combined silver-cholinesterase staining. Colchicine treatment produced abnormalities of innervation pattern characteristic of preterminal collateral sprouting. Because colchicine disrupts axonal transport, we interpret these results to mean that interruption of axonal transport in L4 fibers stimulates intramuscular sprouting of L5 fibers. The data are consistent with Diamonds hypothesis that nerves possess a propensity for collateral growth which is ordinarily repressed by factors that are dependent on axonal transport.

ELECTRON MICROSCOPIC STUDY ON SPECIFIC PROTECTION OF ISOLATED BORDETELLA BRONCHISEPTICA ANTIBODY DURING EXHAUSTIVE LABELLING WITH URANIUM

Exposure of antibody to relatively high concentrations of uranium yielded an electron-dense specific localizing reagent. Destruction of antibody activity during labelling was prevented by protection of the antibody-antigen combining sites: Anti-Bordetella bronchiseptica antibody was removed from serum by absorption with B. bronchiseptica. The washed agglutinate was exposed to uranium. The antibody recovered from this agglutinate by brief alkali treatment in the cold was 80 to 100% pure and contained 28 to 312 atoms of uranium per unit of 156,000 molecular weight. The material stained the walls of living or formalin killed, and the cytoplasm of living B. bronchiseptica. E. coli and mouse spleen cells were not stained. Contrast was diminished when B. bronchiseptica was exposed to unlabelled anti-B. bronchiseptica antiserum prior to uranium-antibody conjugate. Absorption with B. bronchiseptica, but not with E. coli, abolished the staining capacity of purified uraniumn-antibody conjugate. Whole antiserum labelled with uranium without specific protection, even though retaining some antibody activity, was not suitable as a stain since it deposited a large amount of debris and apparently stained both E. coli and B. bronchiseptica.

Functional deficits and anatomical alterations after high cervical spinal hemisection in the rat

Abstract A recent report by Matinian and Andreasian (1976. Enzyme Therapy in Organic Lesions of the Spinal Cord. Brain Information Service, University of California, Los Angeles) concluded that regeneration of the spinal cord sufficient to restore walking occurred in spinal rats (T5 transection) that had been treated by repeated injections of trypsin and hyaluronidase at the site of the lesion. These enzymes were believed to decrease the intense connective tissue and neuroglial scar which otherwise acts as a mechanical barrier to nerve fiber outgrowth. The present experiment tested whether or not these enzymes might similarly facilitate regeneration and functional restitution of the spinal respiratory pathway. The spinal cord of rats was hemisected at C2 (i.e., rostral to the phrenic nucleus), thereby paralyzing the ipsilateral hemidiaphragm. Hyaluronidase and trypsin were injected at the time of operation, and for 15 days thereafter each enzyme was administered on alternate days. Control animals received injection of vehicle. All injections were given subcutaneously at the site of injury. After 5 to 6 months, diaphragmatic function was assessed by electromyography. The spinal cord was then fixed by perfusion with Bouins solution and embedded in paraffin, and sections were prepared and stained for connective tissue fibers, glial fibers, and nerve fibers. None of the treated or control animals showed any evidence of diaphragmatic activity on the operated side. In every animal the site of injury was composed of a dense connective tissue scar surrounded by an encapsulating neuroglial scar. Well-oriented nerve fibers grew to the margin of the lesion where they became deflected. A small number of fibers entered the lesion, but none grew across the injured region. These observations do not support the contention that enzyme treatment facilitates regeneration of the injured spinal cord.

METHOD FOR ENHANCEMENT OF ELECTRON MICROSCOPIC VISUALIZATION OF EMBEDDED ANTIGEN BY BRIDGING OSMIUM TO URANIUM ANTIBODY WITH THIOCARBOHYDRAZIDE

Thiocarbohydrazide (TCH), a bidentate ligand, capable of bridging osmium to osmium in ultrathin sections (OTO method), was shown to possess the additional property of binding osmium to uranium-labeled antibody. On sections in which acrolein-fixed antigen had been localized for electron microscopy by uranium antibody, application of TCH followed by osmication (T-O procedure) greatly enhanced the specific contrast afforded. In addition, the T-O procedure yielded better resolution and better stability to the electron beam than the immunouranium technique employed without this intensification. Even osmium tetroxide-fixed tissue could be used, providing thiosemicarbazide (TSC) was employed to block the later reaction of TCH with tissue-bound osmium and to limit, thereby, the ligation of TCH to the uranium-labeled antibody.