Ken W.S. Ashwell

Advances in Secondary Spinal Cord Injury: Role of Apoptosis

The outcome of spinal cord injury depends on the extent of secondary damage produced by a series of cellular and molecular events initiated by the primary trauma. This article reviews the evidence that secondary spinal cord injury involves the apoptotic as well as necrotic death of neurons and glial cells. Also discussed are the major factors that can contribute to cell death, such as glutamatergic excitotoxicity, free radical damage, cytokines, and inflammation. The development of innovative therapeutic strategies to reduce secondary spinal cord injury depends on an increased understanding of secondary injury mechanisms at the molecular and biochemical level. Such therapeutic interventions may include the use of antiapoptotic drugs, free radical scavengers, and anti-inflammatory agents. These could be targeted to block key reactions on cellular and molecular injury cascades, thus reducing secondary tissue damage, minimizing side effects, and improving functional recovery.

Microglia and cell death in the developing mouse cerebellum.

The appearance and distribution of microglia in the developing cerebellum has been examined with the aid of a peroxidase-conjugated lectin derived from Griffonia simplicifolia. This distribution has in turn been correlated with that of pyknotic figures in the same Nissl-counterstained sections, in order to gain an understanding of the role of microglial in the developing cerebellum. Round and ameboid microglia may be recognised in the fetal cerebellum as early as E11. Numbers of microglia increase steadily from that time, with initial concentrations in the region of the dorsal and ventricular surfaces. By P1, concentrations of both pyknotic figures and ameboid microglia begin to appear in the region of the future cerebellar medulla. Ameboid microglia are recognisable in the cerebellar medulla until P10, with particular concentrations where folia branch and in the rostral cerebellar peduncles. After this time only resting microglia are found in the cerebellum. Concentrations of microglia largely match the positions of pyknotic figures throughout development, except at P10 and P14, when cell death is found in the external granular layer without an accompanying concentration of microglia. Electron microscopic examination of the phagosomes of ameboid microglia at P5 and P6 indicates that these cells are mainly concerned with the phagocytosis of entire cells rather than axons. Cell death in the cerebellar medulla may serve to clear pathways for developing cortical afferents and efferents, or to increase the mechanical plasticity of the medulla during cortical folding.

Trigeminal Sensory System

This chapter addresses the unique anatomy of the pathway for facial sensations, involving the trigeminal ganglion and its associated nuclei within the brainstem. The innervation of specialized cranial structures such as the teeth, tongue, oral and nasal mucosa, cornea, meninges, and conjunctiva are considered. This chapter will also address trigeminal mechanisms in clinically relevant conditions such as toothache, headache and trigeminal neuralgia including using advances in imaging techniques and resolution. Thus it is now possible to obtain functional MR images (fMRI) of the trigeminal pathway from ganglion to cortex. Magnetoencephalography (MEG) and fMRI techniques have provided more details on cortical organization in facial regions of both S1 and S2, while diffusion tensor imaging has been useful for visualizing trigeminothalamic pathways. Plasticity of the system after injury, its association with pain conditions, and the opportunities that this offers for training and rehabilitation, are further areas of current research that are discussed.

THE DISTRIBUTION OF MICROGLIA AND CELL DEATH IN THE FETAL RAT FOREBRAIN

The appearance and distribution of microglia in the fetal and early postnatal rat forebrain have been examined with the aid of a peroxidase-conjugated lectin derived from Griffonia simplicifolia. This distribution has in turn been correlated with that of pyknotic figures in the same Nissl-counterstained sections. Round and ameboid microglia may be recognised in the fetal forebrain as early as E11, at a stage when the telencephalic vesicles are beginning to develop. By E13, concentrations of round microglia are found at the dorsal and rostral limits of the diencephalic vesicle (dorsal lamina terminalis) and in the adjacent medial walls of the telencephalic vesicles. These cells are often seen to have pyknotic material within their cytoplasm. Microglia remain concentrated in this region until E17. From E15, blood vessels and round and ameboid microglia concentrate in the region of the future hippocampus and appear to be drawn into the hippocampal fissure as the cortical plate folds to form Ammons horn. At E15, ameboid microglia are also concentrated in the developing fornix, which first becomes apparent at this age. Microglia remain concentrated in the septomesocortical junction area, and may contribute to the concentrations of microglia previously reported in the region of the developing corpus callosum and cavum septi pellucidi. Microglia probably concentrate in the dorsal lamina terminalis and medial telencephalon at E13 in response to the cell death noted in this region, but other concentrations of microglia in the forebrain are not accompanied by similar aggregations of cell death. These findings indicate that the junction of the telencephalon and rostral diencephalon attracts concentrations of microglia from E13 throughout fetal and early postnatal life, coincident with the infolding of the hippocampus (E13-E19) and several days before the development of the corpus callosum (from E19 onwards).

Dynamics of expression of apoptosis-regulatory proteins Bid, Bcl-2, Bcl-X, Bax and Bak during development of murine nervous system

We have used immunohistochemistry and immunoblotting to examine the expression of Bid and four other Bcl-2 family proteins (Bcl-2, Bcl-X, Bax and Bak) in the developing and adult murine central nervous system (CNS). Bid protein is widespread in embryonic and postnatal brain, and its expression is maintained at a high level late into the adulthood. Bid is expressed both in the germ disc, early neural tube, proliferating stem cells of ventricular zones, and in postmitotic, differentiated neurons of the developing central and peripheral nervous system. As the differentiation proceeds, the neurons express higher levels of Bid than the stem cells of the paraventricular zone. Both in embryonic and postnatal life, Bid protein is present in the most vital regions of brain, such as the limbic system, basal ganglia, mesencephalic tectum, Purkinje cells in cerebellum, and the ventral columns of spinal cord. The p15 cleaved form of Bid was detectable in the brain specimens at fetal stages of development, consistent with caspase-mediated activation of this pro-apoptotic Bcl-2 family protein. Among the Bcl-2 family proteins only Bid and Bcl-XL continue to be expressed at high levels in the adult brain.

Organization of human hypothalamus in fetal development

The organization of the human hypothalamus was studied in 33 brains aged from 9 weeks of gestation (w.g.) to newborn, using immunohistochemistry for parvalbumin, calbindin, calretinin, neuropeptide Y, neurophysin, growth‐associated protein (GAP)‐43, synaptophysin, and the glycoconjugate 3‐fucosyl‐ N‐acetyl‐lactosamine. Developmental stages are described in relation to obstetric trimesters. The first trimester (morphogenetic periods 9–10 w.g. and 11–14 w.g.) is characterized by differentiating structures of the lateral hypothalamic zone, which give rise to the lateral hypothalamus (LH) and posterior hypothalamus. The PeF differentiates at 18 w.g. from LH neurons, which remain anchored in the perifornical position, whereas most of the LH cells are displaced laterally. A transient supramamillary nucleus was apparent at 14 w.g. but not after 16 w.g. As the ventromedial nucleus differentiated at 13–16 w.g., three principal parts, the ventrolateral part, the dorsomedial part, and the shell, were revealed by distribution of calbindin, calretinin, and GAP43 immunoreactivity. The second trimester (morphogenetic periods 15–17 w.g., 18–23 w.g., and 24–33 w.g.) is characterized by differentiation of the hypothalamic core, in which calbindin‐ positive neurons revealed the medial preoptic nucleus at 16 w.g. abutted laterally by the intermediate nucleus. The dorsomedial nucleus was clearly defined at 10 w.g. and consisted of compact and diffuse parts, an organization that was lost after 15 w.g. Differentiation of the medial mamillary body into lateral and medial was seen at 13–16 w.g. Late second trimester was marked by differentiation of periventricular zone structures, including suprachiasmatic, arcuate, and paraventricular nuclei. The subnuclear differentiation of these nuclei extends into the third trimester. The use of chemoarchitecture in the human fetus permitted the identification of interspecies nuclei homologies, which otherwise remain concealed in the cytoarchitecture. J. Comp. Neurol. 446:301–324, 2002.

The development of cranial nerve and visceral afferents to the nucleus of the solitary tract in the rat

We have used carbocyanine dye tracing techniques to examine the distribution of afferents from the facial, trigeminal and vagal nerves to the nucleus of the solitary tract (NST) in the developing rat (E13 to P13). Crystals of DiI (1, 1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate) were placed (unilaterally) into the facial or trigeminal ganglia, or into the cervical vagus nerve, and the sections examined with a laser scanning confocal microscope. Inputs from some peripheral structures (tongue, aortic arch, right atrium and lung) to the NST were also analyzed to provide information on the distribution of organ-specific afferents. No afferents were labeled following DiI placement in the above sites at E13. At E14, a few axons from the geniculate ganglion of the facial nerve were present in the NST anlage, but these were restricted to the area adjacent to the solitary tract. These axons began to invade the medial NST at E15. By E17, facial afferent axons had become widespread throughout rostral NST and from E19 the distribution of DiI labeling displayed a morphologically mature pattern. DiI-labeled afferent axons from the trigeminal nerve first emerged into the NST anlage at E14, initially coursing medially to penetrate the ventricular zone. Between E15 and E17, axonal density increased markedly but after E17 became progressively confined to the lateral NST. Axons from the vagus nerve first appeared in the caudal NST as early as E14 and coursed directly into the proliferative zone of the alar plate at all rostrocaudal levels by E15. From E19 through postnatal life, the distribution of vagal afferent axons was essentially stable with particularly dense label in the caudal NST. Cranial nerve afferents to the NST appear to be distributed to appropriate sites from the beginning of ingrowth, with the exception of trigeminal afferents, where some small initial exuberance was found. The terminal fields derived from selected peripheral organs such as lung, right atrium, aortic arch and tongue were also predominantly distributed to appropriate subnuclei from the beginning of ingrowth into the NST, although organ-specific afferent fields appeared to develop dense arbors somewhat later than did individual cranial nerves. Electron microscopy was used to examine regional synapse development in the rat NST. There was some delay between the ingrowth of afferents to the NST (E15) and the first appearance of synaptic thickenings. The earliest synapses were simple (usually) symmetrical membrane thickenings (from E17) and vesicles did not appear until E19. High synaptic density within the C subnucleus appeared during early postnatal life. Synaptic glomeruli, which are a characteristic feature of afferent input to the adult NST, had not developed by birth, indicating that the pre- and perinatal function of the NST must be mediated through simpler, single, axodendritic inputs to NST neurons.

Organization of the human paraventricular hypothalamic nucleus

The cyto‐ and chemoarchitecture of the human paraventricular hypothalamic nucleus (Pa) was studied with the aid of three‐dimensional computer reconstruction. The adult human Pa is a vertically elongated structure that abuts the wall of the third ventricle (3V) medially and is indented dorsolaterally by the descending fornix. Chemoarchitecture revealed the following five subnuclei in the human Pa. The most prominent of these is the magnocellular subnucleus (PaM) occupying the ventrolateral quadrant of the Pa and comprised of a concentration of large arginin‐vasopressin (AVP)‐ and acetylcholinesterase (AChE)‐positive cells, and small calbindin (Cb)‐positive neurons. Rostrally, the PaM is succeeded by the small anterior parvicellular subnucleus (PaAP), which contains small AChE‐, AVP‐ and tyrosin hydroxylase (TH)‐positive cells. Dorsal to the PaM is found the dorsal subnucleus (PaD), containing large spindle‐shaped TH‐, oxytocin (OXY)‐, and AChE‐positive cells, as well as a population of small Cb‐positive neurons. Abutting the wall of the 3V and medial to PaM and PaD is the parvicellular subnucleus (PaP). The PaP contains small cells immunoreactive for corticotropin‐releasing factor (CRF), neuromedin K receptor (NK3), and nonphosphorylated neurofilament protein (SMI32). The posterior subnucleus (PaPo) is situated posterior to the descending column of the fornix; it replaces all above‐mentioned subdivisions caudally, and is a chemoarchitectonic amalgam that includes dispersed large AChE‐, OXY‐, AVP‐ and TH‐positive cells, as well as small NK3‐, CRF‐, SMI32‐ and Cb‐immunoreactive neurons. The present findings suggest that the human PaM and PaD are homologues to the magnocellular subnuclei of the rat Pa, whereas the human PaP and PaPo correspond to the rat medial parvicellular and posterior subnuclei, respectively. J. Comp. Neurol. 423:299–318, 2000.

Olfactory ensheathing cells: their potential use for repairing the injured spinal cord.

Study Design. The literature concerning the potential use of olfactory ensheathing cells for repairing damaged spinal cord was reviewed. Objective. To engender a better understanding of the role that olfactory ensheathing cells play in spinal cord regeneration. Summary of Background Data. Intraspinal transplants (e.g., fetal neuronal cells, progenitor stem cells, and olfactory ensheathing cells) have been used to restore intraspinal circuitry or to serve as a “bridge” for damaged axons. Among these transplants, olfactory ensheathing cells provide a particularly favorable substrate for spinal axonal regeneration because these cells can secrete extracellular molecules and neurotrophic factors and have the ability to migrate into gliotic scar tissue, an important attribute that might be associated with high potential for axonal regeneration. Methods. Recent advances using centrally and peripherally derived olfactory ensheathing cells to promote spinal cord regeneration were reviewed. Results. Both centrally and peripherally derived olfactory ensheathing cells can lead to a degree of functional and anatomic recovery after spinal cord injury in adult animals. Conclusion. Olfactory ensheathing cells from olfactory lamina propria in the nose are among the best transplants for “bridging” descending and ascending pathways in damaged spinal cord.

Is There a Zone of Vascular Vulnerability in the Fetal Brain Stem

The pattern of malformations in congenital anomalies such as Möbius syndrome and following prenatal cocaine exposure suggests that there is a zone of vascular vulnerability or ischemic sensitivity in the paramedian region of the developing brain stem. In the present study, postmortem examination of the brain of an infant with Möbius syndrome revealed mineralized foci concentrated in paramedian wedge-shaped areas of the pontine and medullary tegmentum. We also examined the development of brain stem vasculature in the rat at the light and ultrastructural level to determine whether anatomical features of the paramedian brain stem region could contribute to elevated incidence of vascular accidents in that zone. Several observations of relevance to the question of vascular vulnerability of the midline were made. Firstly, and as previously noted by other authors, the brain stem midline remains avascular for protracted periods during fetal life. We propose that the inability of vessels in the paramedian region to anastomose across the avascular midline gives rise to paramedian watershed zones that could be vulnerable to ischaemia in the event of hypoperfusion due to teratogenic action. Secondly, we studied the development of cytochrome oxidase activity in the fetal brain stem and noted high oxidative metabolic activity of the somatic efferent nuclei in the paramedian region, which could render their constituent neurons particularly susceptible to hypoxia. Thirdly, our ultrastructural examination revealed large amounts of extracellular space surrounding paramedian pontine vessels in comparison to laterally placed vessels, although there was no significant difference between the vessels of the two regions in tight junction length and endothelial thickness. We propose that the greater proportion of unoccupied extracellular space surrounding medial vessels may contribute to poorer support of these vessels in ischemic/reperfusion episodes. This poor support could in turn give rise to an increased risk of hemorrhage.