Joel A. Black

Primary cortical motor neurons undergo apoptosis after axotomizing spinal cord injury.

Spinal cord injury (SCI) results in loss of voluntary motor control followed by incomplete recovery, which is partly mediated by the descending corticospinal tract (CST). This system is an important target for therapeutic repair strategies after SCI; however, the question of whether apoptotic cell death occurs in these axotomized neurons remains unanswered. In this study, adult (150–175 g) male Sprague‐Dawley rats underwent T9 transection of the dorsal funiculus, which axotomizes the dorsal CST, and introduction of the retrograde tracer Fluoro‐Gold into the lesion site. Primary motor cortex (M1) was then examined for evidence of apoptosis weekly for 4 weeks after injury. Axotomized pyramidal cells, identified by retrograde transport of Fluoro‐Gold, were found in M1 (57.5 ± 9.6/median section, 6,127 ± 292 total), and a significant proportion were terminal deoxynucleotidyl transferase (TdT) ‐mediated deoxyuridine triphosphate (dUTP)‐rhodamine nick end labeling (TUNEL) ‐positive at 1 week after injury (39.3 ± 5.6%), compared with animals undergoing sham surgery (1.2 ± 1.4%). At 2–4 weeks, fewer cells were Fluoro‐Gold–positive (24.6 ± 65.06 to 25.3 ± 6.4/median section, 2,338 ± 233 to 2,393 ± 124 total), of which very few were TUNEL‐positive. In TUNEL‐positive cells, Hoechst 33342 staining revealed nuclear morphology consistent with apoptosis, chromatin condensation, and formation of apoptotic bodies. Fluoro‐Gold–positive cells showed increased caspase‐3 and Bax immunoreactivity. Hematoxylin and eosin staining revealed similar nuclear changes and dystrophic cells. Internucleosomal DNA fragmentation was detected by gel electrophoresis at the 1‐week time point. Lesioned animals not receiving Fluoro‐Gold exhibited the same markers of apoptosis. These results document, for the first time, features of apoptotic cell death in a proportion of axotomized cortical motor neurons after SCI, suggesting that protection from apoptosis may be a prerequisite for regenerative approaches to SCI. J. Comp. Neurol. 462:328–341, 2003.

Protection of the axonal cytoskeleton in anoxic optic nerve by decreased extracellular calcium

Since CNS white matter tracts contain axons, oligodendrocytes and astrocytes but not synapses, it is likely that anoxic injury of white matter is mediated by cellular mechanisms that do not involve synapses. In order to test the hypothesis, that anoxic injury of white matter is mediated by an influx of Ca2+ into the intracellular compartment of axons, we compared the ultrastructure of axons in rat optic nerve exposed to 60 min of anoxia in artificial cerebrospinal fluid (aCSF) containing normal (2 mM) Ca2+, and in aCSF containing zero-Ca2+ together with 5 mM EGTA. Optic nerves fixed at the end of 60 min of anoxia in 2 mM Ca2+ exhibit extensive ultrastructural alterations including disruption of microtubules and neurofilaments within the axonal cytoskeleton, development of membranous profiles and empty spaces between the axon and the ensheathing myelin, and swelling of mitochondria with loss of cristae. Bathing the nerves in zero-Ca2+ aCSF during anoxia protected the axons from cytoskeletal changes; after 60 min of anoxia, optic nerve axons retained normal-appearing microtubules and neurofilaments. Membranous profiles were rare, and empty spaces between axons and myelin did not develop in anoxic optic nerves bathed in zero-Ca2+ aCSF. Disorganization of cristae in axonal mitochondria was observed in anoxic optic nerves even when Ca2+ was omitted from the medium. Because Ca(2+)-mediated injury is known to disrupt the axonal cytoskeleton, these results support the hypothesis that anoxia triggers an abnormal influx of Ca2+ into myelinated axons in CNS white matter.

Schwann Cell Glycogen Selectively Supports Myelinated Axon Function

Interruption of energy supply to peripheral axons is a cause of axon loss. We determined whether glycogen was present in mammalian peripheral nerve, and whether it supported axon conduction during aglycemia.

Freeze-fracture ultrastructure of rat C.N.S. and P.N.S. nonmyelinated axolemma

SummaryThe axolemma of nonmyelinated fibres from the corpus callosum and cerebellar cortex (C.N.S.) and the vagus nerve (P.N.S.) was investigated with freeze-fracture electron microscopy. The major observations of this study are as follows: (1) there is a highly asymmetrical distribution of intramembranous particles between the E- and P-fracture faces in both C.N.S. and P.N.S. fibres; (2) the total number of particles on the P-faces of all axonal types studied is considerably greater than that on the E-face; (3) the number of particles on the E-faces of C.N.S. axons is greater than that on the E-faces of P.N.S. axons; and (4) the percentage of large (>9.6 nm) particles is greater on the E-face than on the P-face regardless of the axon studied. The results are compared with previous freeze-fracture investigations on the nodal and internodal membranes of myelinated fibres.

Perinodal astrocytic processes at nodes of ranvier in developing normal and glial cell deficient rat spinal cord

This study examined, during normal development and during the development of a glial cell deficient axon population, the nature of astrocyte involvement at the central nodes of Ranvier on spinal cord axons. One condition examined was the ventral funiculus of normal 7-day-old rats. At this age, the lumbar spinal cord underwent an active phase of gliogenesis, and axons were seen in various stages of myelination. Perinodal astrocytic processes were routinely observed at nodes of axons on which myelin sheaths exceeded 8 compact lamellae. Perinodal astrocytic processes were also seen in close proximity to axolemma at most developing nodes. This study also examined the lumbar spinal cords of rats which were X-irradiated on the third postnatal day. This procedure caused a profound reduction in the astrocyte and oligodendrocyte population in 13- and 18-day-old rats, while sparing the neuronal elements. Thus, axo-glial relationships observed in this tissue are unlikely to be random occurrences. Despite the reduction in glial cells, some oligodendrocyte-myelinated axons were observed in the irradiated spinal cords. Perinodal astrocytes were seen at all oligodendrocyte-derived nodes observed in the irradiated cord and appeared to have a specific relationship to the node of Ranvier. The presence of astrocytic processes at the normal, developing node and at the nodes in glial cell deficient spinal cords suggests that astrocytes may be necessary to the function of nodal axolemma. In irradiated spinal cords, where the glial cells are markedly reduced, apposition between astrocytic and oligodendrocytic membrane at the paranode and internode was also seen and was so common that it is highly unlikely to be due to random occurrences. These observations further suggest that in addition to the presumptive role at the nodes, astrocytes may play an inductive or supportive role in the development and maintenance of central myelin.

Membrane specialization and axo-glial association in the rat retinal nerve fibre layer: freeze-fracture observations

SummaryThe ultrastrucrure of non-myelinated ganglion cell axolemma within the retinal nerve fibre layer of adult rats was examined by thin section and freeze-fracture electron microscopy. Most of the axolemma within the nerve fibre layer does not exhibit any membrane specializations; intramembranous particles are partitioned with a density of ∼1750 μm−2 on the P-fracture face and ∼225 μm−2 on the E-face of the non-specialized axolemma. The nerve fibres also exhibit specialized foci of axolemma, at which the axons are abutted by the tips of blunt, radially oriented processes from Müller cells. At such sites of axo-glial association, an electron-dense undercoating is present beneath the axon membrane. Freeze-fracture analysis revealed a substantial increase in the density of E-face particles (>500 μm−2) at sites of association between the tips of blunt glial processes and the axon. These findings demonstrate that non-myelinated axolemma of the retinal nerve fibre layer can exhibit spatial heterogeneity, with patches of node-like membrane at regions of specialized association with glial cell processes. On the basis of their morphological similarity to nodes of Ranvier, we suggest that these specialized axon regions represent foci of inward ionic current.

Membrane ultrastructure of developing axons in glial cell deficient rat spinal cord

SummaryIn order to investigate axolemmal development in a glial cell deficient environment, normal and irradiated dorsal funiculus in rat lumbosacral spinal cord was examined by freeze-fracture electron microscopy. At 3 days of age, normal fibres are all unmyelinated and of small (<0.5 μm) diameter. The unmyelinated axons have a moderate density (∼850 μm−2) of intramembranous particles (IMPs) on P-fracture faces and a low IMP density (∼300 μm−2) on E-faces. IMPs are homogeneously distributed along both fracture faces. By 19 days of age, the normal dorsal funiculus is well populated with myelinated axons and glial cells, as well as a sizable population of unmyelinated fibres. Nearly all of the myelinated fibres have a large (>1.0 μm) diameter; whereas, most unmyelinated axons are of small (<0.5 μm) calibre. The axolemma of unmyelinated axons is relatively undifferentiated, with an asymmetrical distribution of IMPs (P-face: ∼1100 μm−2; E-face: ∼450 μm−2). Myelinated fibres show nodal and paranodal regions with P-face and E-face ultrastructure similar to previous descriptions. Internodal axolemma appears relatively homogeneous, with P-faces being highly particulate (∼2100 μm−2) and a low IMP density (∼200 μm−2) on E-faces. Following irradiation of the lumbosacral spinal cord at 3 days of age, there is a severe reduction in the number of glial cells and myelinated fibres in this region when the tissue is examined at 19 days of age. Despite the deficiency of glial cells in this tissue, axonal and axolemmal development continue. Numerous large (>1.0 μm) diameter axons are present in this irradiated tissue. Large diameter axons show a high (∼2000 μm−2) density of IMPs on P-faces; E-face IMP density remains at ∼440 μm−2. Small calibre axons also have an asymmetrical distribution of particles (P-face: ∼1100 μm−2; E-face: 280 μm−2). The axolemmal E-faces of some glial cell deprived fibres exhibit regions with greater than normal (∼750 μm−2) density of IMPs. These results demonstrate that some aspects of axonal and axolemmal development continue in a glial cell deficient environment, and it is suggested that axolemmal ultrastructure is, at least in part, independent of glial cell association.

Carbonic anhydrase activity develops postnatally in the rat optic nerve.

We examined the appearance of carbonic anhydrase (CA) activity in rat optic nerves (RONs) 5-77 postnatal days of age and correlated the appearance of enzyme activity with structural and physiological alterations. CA activity was nearly absent before 10 days of age and appeared in this CNS white matter tract with a developmental time-course similar to that of oligodendrogliogenesis and myelinogenesis. When oligodendrocytes and myelin were depleted in the RON by treatment with a mitotic inhibitor, CA activity was markedly reduced. These observations support the hypothesis that CA is contained primarily in oligodendrocytes and myelin. Neural activity in the RON caused changes in extracellular pH (pHo) and the character of these pHo responses was very age dependent; older nerves exhibited much larger acid shifts than neonatal nerves. The development of CA activity may be a factor contributing to this physiological alteration.

Nav1.7 is the predominant sodium channel in rodent olfactory sensory neurons

BackgroundVoltage-gated sodium channel Nav1.7 is preferentially expressed in dorsal root ganglion (DRG) and sympathetic neurons within the peripheral nervous system. Homozygous or compound heterozygous loss-of-function mutations in SCN9A, the gene which encodes Nav1.7, cause congenital insensitivity to pain (CIP) accompanied by anosmia. Global knock-out of Nav1.7 in mice is neonatal lethal reportedly from starvation, suggesting anosmia. These findings led us to hypothesize that Nav1.7 is the main sodium channel in the peripheral olfactory sensory neurons (OSN, also known as olfactory receptor neurons).MethodsWe used multiplex PCR-restriction enzyme polymorphism, in situ hybridization and immunohistochemistry to determine the identity of sodium channels in rodent OSNs.ResultsWe show here that Nav1.7 is the predominant sodium channel transcript, with low abundance of other sodium channel transcripts, in olfactory epithelium from rat and mouse. Our in situ hybridization data show that Nav1.7 transcripts are present in rat OSNs. Immunostaining of Nav1.7 and Nav1.6 channels in rat shows a complementary accumulation pattern with Nav1.7 in peripheral presynaptic OSN axons, and Nav1.6 primarily in postsynaptic cells and their dendrites in the glomeruli of the olfactory bulb within the central nervous system.ConclusionsOur data show that Nav1.7 is the dominant sodium channel in rat and mouse OSN, and may explain anosmia in Nav1.7 null mouse and patients with Nav1.7-related CIP.

A quantitative study of developing axons and glia following altered gliogenesis in rat optic nerve

Axonal and glial cell development within rat optic nerve in which gliogenesis was altered by systemic injection of 5-azacytidine (5-AZ) was examined by quantitative electron microscopy. In neonatal (0-2 days) rat optic nerves, all fibers are premyelinated, and they exhibit a fairly uniform diameter (approximately 0.22 micron). These fibers occupy approximately 55% of the optic nerve volume. At this early age, glia within the optic nerve consist only of cells of astrocytic lineage and progenitor cells. These glia occupy approximately 28% of the optic nerve volume, and there are approximately 80 glial cells/optic nerve cross section. In 14-day-old normal optic nerves, myelinated and ensheathed fibers comprise approximately 17% and 9%, respectively, of the total number of axons. Mean axonal diameter of myelinated fibers is approximately 0.75 micron, while mean diameter for ensheathed axons is approximately 0.50 micron. By volume, these fibers occupy approximately 25% of the nerve, which is similar to the volume occupied by premyelinated axons in these nerves. At 14 days of age, there are approximately 300 glial cells/optic nerve transverse section, and these glia occupy approximately 37% of the volume in normal optic nerve. Oligodendroglia represent approximately 40% of total glial cells present, while astroglia and progenitor cell each comprise approximately 30% of the cells. In optic nerves from 14-day-old rats treated with 5-AZ, few myelinated fibers are present and the number of oligodendroglia is markedly reduced. Axonal diameter of premyelinated fibers is similar to that of age-matched controls. Myelinated and ensheathed fibers comprise approximately 2% of the total fibers present in 5-AZ-treated optic nerves, with the remaining fibers being premyelinated. The few myelinated and ensheathed fibers present in 5-AZ-treated optic nerves display similar axonal diameters to corresponding fibers from age-matched control tissue. Glial cells occupy approximately 40% of the nerve volume, and there are approximately 200 glia/nerve cross section in 5-AZ-treated rats. Astroglia comprise approximately 63% of the total glial cells, while approximately 12% of the cells are oligodendroglia. These results demonstrate that 5-AZ is a potent inhibitor of oligodendrogliogenesis, with a concomitant marked reduction in the number of myelinated fibers.