Henry C. Powell

Nerve regeneration in silicone chambers: Influence of gap length and of distal stump components

Abstract The range of growth-promoting influences from a distal nerve stump on a regenerating proximal stump was determined using an experimental system in which a gap between cross-anastomosed rat sciatic nerves was encased by a cylindrical silicone chamber. Two arrangements were examined after 1 month in situ: A proximal-distal (PD) system in which both proximal and distal stumps were introduced into the ends of the chamber, and a proximal-open (PO) system in which the distal stump was omitted. When the gap was 6 mm long, a regenerated nerve extended all the way through the chamber in both the PD and PO systems. When the gap was increased to 10 mm, a similar regrowth occurred in the PD chamber, whereas in the PO chamber proximal regrowth was partial or nonexistent. When the gap was increased to 15 mm, no regeneration occurred, even in the presence of the distal stump. These observations confirm that the distal stump influences proximal regeneration and indicate that this influence can act only over a limited distance or volume. Such an influence could consist of humoral agents which support nerve growth and/or outgrowth from the distal stump.

In vivo regeneration of cut nerves encased in silicone tubes: growth across a six-millimeter gap.

We describe an experimental in vivo system for studying peripheral nerve regeneration, in which the proximal stump of a transected nerve regrows through a transparent silicone chamber toward the distal stump. Physical separation permits examination of the effects of the humoral and/or cellular influences from the distal stump on regenerating fibers before they invade the distal segment itself. A small segment of the rat sciatic nerve was resected, leaving a 6 mm gap which was then encased by a cylindrical silicone chamber. Within the first weeks, a nerve trunk regenerated along the central axis of the chamber bridged the gap between the proximal and distal stumps. When the distal nerve stump was omitted from the distal opening of the chamber, only a thin structure with a few small-caliber fibers extended across the gap. In each instance regenerating nerve appeared as a cord-like structure completely surrounded by clear fluid, a feature which permits easy collection of the extracellular fluid for analysis of its chemical properties and biological activity. This feature also allows in vivo manipulation of the humoral environment in which nerve regeneration occurs.

Evolution of Neuropathologic Abnormalities Associated with Blood-Brain Barrier Breakdown in Transgenic Mice Expressing Interleukin-6 in Astrocytes

Abstract. As both astrocytes and cytokines modulate the permeability of cerebral endothelial cells, transgenic animal models which overexpress cytokines, such as interleukin-6 (IL-6), may provide insight into the neuropathological consequences of increased BBB permeability. In this study, a GFAP-IL6 transgenic mouse model and horseradish peroxidase (HRP) were used to investigate BBB permeability and associated neuropathologic changes. In the cerebellum of control mice, the BBB developed between postnatal days 7 and 14. In transgenic mice, the BBB never developed and extensive breakdown was evident in both high- and low-expressor animals by 1 month after birth. Vascular proliferation was apparent from birth in association with development and retention of normal cerebellar architecture until 3 and 6 months in high- and low-expressor animals, respectively. At these times, a leptomeningeal inflammatory infiltrate, vacuolated astrocytic foot processes and endothelial abnormalities were apparent in the cerebellum. At 6 months in high-expressor and 12 months in low-expressor animals, parenchymal inflammation, gliosis, spongiform change, axonal degeneration and macrophage accumulation were evident. The findings suggest that increased production of IL-6 can influence the development and physiologic function of the BBB as well as contribute to parenchymal central nervous system injury.

Grafts of genetically modified Schwann cells to the spinal cord: survival, axon growth, and myelination.

Schwann cells naturally support axonal regeneration after injury in the peripheral nervous system, and have also shown a significant, albeit limited, ability to support axonal growth and remyelination after grafting to the central nervous system (CNS). It is possible that Schwann cell-induced axonal growth in the CNS could be substantially increased by genetic manipulation to secrete augmented amounts of neurotrophic factors. To test this hypothesis, cultured primary adult rat Schwann cells were genetically modified using retroviral vectors to produce and secrete high levels of human nerve growth factor (NGF). These cells were then grafted to the midthoracic spinal cords of adult rats. Findings were compared to animals that received grafts of nontransduced Schwann cells. Spinal cord lesions were not placed prior to grafting because the primary aim of this study was to examine features of grafted Schwann cell survival, growth, and effects on host axons. In vitro prior to grafting, Schwann cells secreted 1.5+/-0.1 ng human NGF/ml/10(6) cells/day. Schwann cell transplants readily survived for 2 wk to 1 yr after in vivo placement. Some NGF-transduced grafts slowly increased in size over time compared to nontransduced grafts; the latter remained stable in size. NGF-transduced transplants were densely penetrated by primary sensory nociceptive axons originating from the dorsolateral fasciculus of the spinal cord, whereas control grafts showed significantly fewer penetrating sensory axons. Over time, Schwann cell grafts also became penetrated by TH- and DBH-labeled axons of putative coerulospinal origin, unlike control cell grafts. Ultrastructurally, axons in both graft types were extensively myelinated by Schwann cells. Grafted animals showed no changes in gross locomotor function. In vivo expression of the human NGF transgene was demonstrated for periods of at least 6 m. These findings demonstrate that primary adult Schwann cells 1) can be transduced to secrete augmented levels of neurotrophic factors, 2) survive grafting to the CNS for prolonged time periods, 3) elicit robust growth of host neurotrophin-responsive axons, 4) myelinate CNS axons, and 5) express the transgene for prolonged time periods in vivo. Some grafts slowly enlarge over time, a feature that may be attributable to the propensity of Schwann cells to immortalize after multiple passages. Transduced Schwann cells merit further study as tools for promoting CNS regeneration.

Neurotoxicity of Local Anesthetics: Altered Perineurial Permeability, Edema, and Nerve Fiber Injury

A quantitative, in situ experimental method was developed employing the rat sciatic nerve to study the neurotoxicity of local anesthetic solutions applied directly to an intact peripheral nerve bundle. One-milliliter volumes of 2-chloroprocaine, 3%; tetracaine, 1 %; lidocaine, 2%; bupivacaine, 0.75%; or sodium chloride, 0.2%; were injected with a 30-gauge needle beneath the mesoneurium but exterior to the epineurium. The wound was closed and the animals were normally maintained until the nerves were reexposed for quantitative biophysical and morphologic testing 24 h to 4 weeks later. The results indicate that topically applied 2-chloroprocaine and tetracaine produce significant endoneurial edema 48 h after treatment. Horseradish peroxidase was used to verify increased permeability of the perineurium. Endoneurial fluid pressure was significantly increased in edematous nerves. Electron microscopy revealed abnormal mast cells and proliferation of endoneurial fibro-blasts in addition to Schwann cell injury and axonal dystrophy. This study shows that extrafascicular administration of clinically used concentrations of local anesthetic solutions can alter perineurial permeability, producing changes in the endoneurial environment that are associated with neurotoxic injury. Perineurial and endoneurial fibrolic changes may be a late consequence of peripheral nerve injury with anesthetic solutions producing altered perineurial permeability with endoneurial edema.

Competence of nerve tissue as distal insert promoting nerve regeneration in a silicone chamber.

A new peripheral nerve forms across a 10 mm gap within a silicone chamber regeneration model when the distal segment of a transected sciatic nerve, connected to its end organs, is sutured into the distal end of the chamber. We have tested the ability of other tissue inserts to support axonal regeneration in the chamber. When an isolated 2 mm piece of sciatic nerve was sutured into the distal end, fibrin matrix formation, cell immigration and axonal regeneration were identical to those occurring in the control. When the distal nerve insert was replaced with a 2 mm piece of skin or a ligation, a matrix did not form and subsequent cell immigration and axonal regeneration did not occur. When a 2 mm piece of tendon was inserted, a matrix did form at 1 week, but a structure across the gap was observed at later time periods in only 2 out of 7 chambers. The matrix either dissolved before cells could enter the chamber or did not promote cellular immigration and subsequent axonal regeneration. When the distal end was left open, a matrix formed and cells from the reactive tissue outside the chamber entered the matrix and formed a granulation tissue bridge across the gap. This tissue failed to support axonal regeneration; at 3 weeks, axons stopped 1 mm beyond the proximal stump at the interface with the granulation tissue. Thus, matrix formation and a cellular bridge are necessary but not sufficient to ensure regeneration. Successful regeneration across the silicone chamber gap requires humoral and/or cellular contributions available from peripheral nervous tissue and not from the other tested tissues.

Pressure increase in the dorsal root ganglion following mechanical compression. Closed compartment syndrome in nerve roots

Spinal nerve roots including the dorsal root ganglion (DRG) often are mechanically deformed in connection with degenerative and traumatic conditions of the spine. However, the pathophysiology underlying various functional changes, including pain production, in such conditions is incompletely known. In this study, the tissue fluid pressure in the DRG of L5 nerve roots of rats was measured before and after compression. Normal values were found to be 3.7 ± 0.3 cm H2O (2.7 ± 0.2 mm Hg). After mechanical compression, the endoneurial fluid pressure in the ganglia rose to 9.6 ± 1.7 cm H2O (7.1 ± 1.2 mm Hg) (P < 0.001). Histologic examination revealed edema and haemorrhage in the endoneurial space of the DRG. Pressure increase in the DRG as a result of mechanical deformation by, for example, a herniated disc might be expected to reduce blood flow to the sensory nerve cell bodies in the DRG. This may be a mechanism underlying the production of nerve root pain, which previously has not been described.

Structural and functional neuropathology in transgenic mice with CNS expression of IFN-α1

Abstract Cytokines belonging to the type I interferon (e.g. interferon-α) family are important in the host response to infection and may have complex and broad ranging actions in the central nervous system (CNS) that may be beneficial or harmful. To better understand the impact of the CNS expression of the type I interferons (IFN), transgenic mice were developed that produce IFN-α1 chronically from astrocytes. In two independent transgenic lines with moderate and low levels of astrocyte IFN-α mRNA expression respectively, a spectrum of transgene dose- and age-dependent structural and functional neurological alterations are induced. Structural changes include neurodegeneration with loss of cholinergic neurons, gliosis, angiopathy with mononuclear cell cuffing, progressive calcification affecting basal ganglia and cerebellum and the up-regulation of a number of IFN-α-regulated genes. At a functional level, in vivo and in vitro electrophysiological studies revealed impaired neuronal function and disturbed synaptic plasticity with pronounced hippocampal hyperexcitability. Severe behavioral alterations were also evident in higher expressor GFAP-IFNα mice which developed fatal seizures around 13 weeks of age precluding their further behavioral assessment. Modest impairments in discrimination learning were measured in lower expressor GFAP-IFNα mice at various ages (7–42 weeks). The behavioral and electrophysiological findings suggest regional changes in hippocampal excitability which may be linked to abnormal calcium metabolism and loss of cholinergic neurons in the GIFN mice. Thus, these transgenic mice provide a novel animal model in which to further evaluate the mechanisms that underlie the diverse actions of type I interferons in the intact CNS and to link specific structural changes with functional impairments.

Spinal p38β isoform mediates tissue injury‐induced hyperalgesia and spinal sensitization

Antagonist studies show that spinal p38 mitogen‐activated protein kinase plays a crucial role in spinal sensitization. However, there are two p38 isoforms found in spinal cord and the relative contribution of these two to hyperalgesia is not known. Here we demonstrate that the isoforms are distinctly expressed in spinal dorsal horn: p38α in neurons and p38β in microglia. In lieu of isoform selective inhibitors, we examined the functional role of these two individual isoforms in nociception by using intrathecal isoform‐specific antisense oligonucleotides to selectively block the expression of the respective isoform. In these rats, down‐regulation of spinal p38β, but not p38α, prevented nocifensive flinching evoked by intraplantar injection of formalin and hyperalgesia induced by activation of spinal neurokinin‐1 receptors through intrathecal injection of substance P. Both intraplantar formalin and intrathecal substance P produced an increase in spinal p38 phosphorylation and this phosphorylation (activation) was prevented when spinal p38β, but not p38α, was down‐regulated. Thus, spinal p38β, probably in microglia, plays a significant role in spinal nociceptive processing and represents a potential target for pain therapy.

Late-Onset Chronic Inflammatory Encephalopathy in Immune-Competent and Severe Combined Immune-Deficient (SCID) Mice with Astrocyte-Targeted Expression of Tumor Necrosis Factor

To examine the role of tumor necrosis factor (TNF)-alpha in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-alpha was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-alpha fusion gene. In two independent GFAP-TNFalpha transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25-) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of alpha- and beta-chemokines was induced or upregulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFalpha mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFalpha mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-alpha in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury.