Christopher B. McBride

Minocycline Treatment Reduces Delayed Oligodendrocyte Death, Attenuates Axonal Dieback, and Improves Functional Outcome after Spinal Cord Injury

Minocycline has been demonstrated to be neuroprotective after spinal cord injury (SCI). However, the cellular consequences of minocycline treatment on the secondary injury response are poorly understood. We examined the ability of minocycline to reduce oligodendrocyte apoptosis, microglial/macrophage activation, corticospinal tract (CST) dieback, and lesion size and to improve functional outcome after SCI. Adult rats were subjected to a C7-C8 dorsal column transection, and the presence of apoptotic oligodendrocytes was assessed within the ascending sensory tract (AST) and descending CST in segments (3-7 mm) both proximal and distal to the injury site. Surprisingly, the numbers of dying oligodendrocytes in the proximal and distal segments were comparable, suggesting more than the lack of axon-cell body contiguity played a role in their demise. Minocycline or vehicle control was injected into the intraperitoneal cavity 30 min and 8 hr after SCI and thereafter twice daily for 2 d. We report a reduction of apoptotic oligodendrocytes and microglia within both proximal and distal segments of the AST after minocycline treatment, using immunostaining for active caspase-3 and Hoechst 33258 staining in combination with cell-specific markers. Activated microglial/macrophage density was reduced remote to the lesion as well as at the lesion site. Both CST dieback and lesion size were diminished after minocycline treatment. Footprint analysis revealed improved functional outcome after minocycline treatment. Thus, minocycline ameliorates multiple secondary events after SCI, rendering this clinically used drug an attractive candidate for SCI treatment trials.

Axotomy abolishes NeuN expression in facial but not rubrospinal neurons

The neuronal nuclei (NeuN) antibody, which binds to a poorly characterized antigen/antigens, is increasingly being used in several areas of study as a specific marker to identify neuronal populations. Despite the increasing reliance on NeuN as a panneuronal marker, changes of NeuN expression following axonal injury have not yet been examined. In the present study, NeuN immunoreactivity was analyzed in adult rodent facial motoneurons [peripheral nervous system (PNS) model] following nerve resection or crush and in rubrospinal neurons [central nervous system (CNS) model] after lesion of the dorsal lateral funiculus at the cervical level of the spinal cord. Peripheral nerve resection in the rat and mouse resulted in an almost complete loss of NeuN immunoreactivity in facial motoneurons by 3 days postinjury and remained absent at 28 days post-resection despite the survival of the neurons as evidenced by neuronal tracing. These results were confirmed with Western blot. In the peripheral nerve crush model of injury, there was an initial decline in NeuN immunoreactivity in facial motoneurons, but unlike the resection model, NeuN immunoreactivity began to return within 7 days postinjury and returned to the uninjured level of expression by 28 days. In contrast, axotomy in the CNS model resulted in little decline in NeuN immunoreactivity in the rubrospinal neurons, even after 28 days postaxotomy. These results indicate that NeuN expression in response to axonal injury is different in separate neuronal populations (PNS and CNS), and that care must be taken when addressing cell survival based on NeuN staining alone.

Caspase-3 is activated following axotomy of neonatal facial motoneurons and caspase-3 gene deletion delays axotomy-induced cell death in rodents

In this report, we examined the possible functions of the cell death protease, caspase‐3, in the axotomy‐induced apoptosis of facial motoneurons in newborn rodents. Using in situ hybridization and Western blot, we found higher levels of caspase‐3 mRNA and pro‐caspase‐3 protein expression in motoneurons of neonatal and 2‐week‐old rats than adult rats. Following facial motoneuron axotomy, caspase‐3 mRNA and protein expression increased in motoneurons of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase‐3 protease, we found that catalytically active caspase‐3 was present only in axotomized neonatal motoneurons. As motoneurons in neonatal but not adult rodents are susceptible to axotomy‐induced apoptosis, we hypothesized that caspase‐3 may play a role in their demise. To determine the necessity of caspase‐3 activation in axotomy‐induced apoptosis, we counted the number of surviving motoneurons at 4 and 7 days following axotomy in wild type mice and caspase‐3 gene‐deleted mice. There were nearly three times more surviving motoneurons in caspase‐3 gene‐deleted mice than in wild type mice at both 4 days (mean 1074 vs. 464, P < 0.005) and 7 days (mean 469 vs. 190, P < 0.005) following injury, indicating a slower rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) revealed incomplete nuclear condensation in caspase‐3‐deficient motoneurons. These results demonstrate that caspase‐3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.

Chapter 20 Permissive and restrictive periods for brainstem-spinal regeneration in the chick

Publisher Summary This chapter focuses on the permissive and restrictive periods for brainstem-spinal regeneration in the chick. Studies of the development and organization of brainstem and spinal locomotor mechanisms indicate that birds are very similar to all other vertebrates. For example, the anatomical, physiological, and pharmacological organization of avian brainstem-spinal locomotor circuits is analogous to that of all other vertebrates, including mammal. Moreover, after a complete or incomplete spinal cord injury, adult birds suffer the same motor deficits as adult mammals. The chapter discusses an approach for the search of the cellular factors that underlie the transition between the permissive and restrictive periods for functional regeneration using a number of different techniques. It examines whether plasma membrane fractions, isolated from spinal cord tissue at different stages of development, were permissive or restrictive substrates for the in vitro differentiation and outgrowth of processes from membrane fractions, isolated from spinal cord immortalized neuronal-glial hybrid cell.

Caspase inhibition attenuates transection-induced oligodendrocyte apoptosis in the developing chick spinal cord.

A developmental model of spinal cord injury in the embryonic chick was specifically developed to characterize the involvement of caspases in injury-induced oligodendrocyte apoptosis remote from the lesion and the ability of caspase inhibitors to attenuate this process. Developmental apoptosis in the cervical spinal cord increased within the white matter between embryonic days 13 and 18, the period of myelination of this region. Spinal cord transection during this period induced a rapid increase in apoptotic cells in the ventral and lateral white matter over several millimeters caudal to the injury. Immunostaining identified large numbers of these cells as oligodendrocytes. Catalytic activity assays and immunostaining demonstrated caspase-3-like but not caspase-1-like activity to be involved in this apoptotic response. In vivo application of specific caspase inhibitors significantly attenuated transection-induced apoptosis. Thus, we describe a developmental period during which spinal oligodendrocytes exhibited a heightened, caspase-dependent sensitivity to transection-induced apoptosis that is attenuated by caspase inhibition.

Evidence-based scientific exercise guidelines for adults with spinal cord injury: an update and a new guideline

ObjectivesTo describe the process and outcomes of using a new evidence base to develop scientific guidelines that specify the type and minimum dose of exercise necessary to improve fitness and cardiometabolic health in adults with spinal cord injury (SCI).SettingInternational.MethodsUsing Appraisal of Guidelines, Research and Evaluation (AGREE) II reporting criteria, steps included (a) determining the guidelines’ scope; (b) conducting a systematic review of relevant literature; (c) holding three consensus panel meetings (European, Canadian and International) to formulate the guidelines; (d) obtaining stakeholder feedback; and (e) process evaluation by an AGREE II consultant. Stakeholders were actively involved in steps (c) and (d).ResultsFor cardiorespiratory fitness and muscle strength benefits, adults with a SCI should engage in at least 20 min of moderate to vigorous intensity aerobic exercise 2 times per week AND 3 sets of strength exercises for each major functioning muscle group, at a moderate to vigorous intensity, 2 times per week (strong recommendation). For cardiometabolic health benefits, adults with a SCI are suggested to engage in at least 30 min of moderate to vigorous intensity aerobic exercise 3 times per week (conditional recommendation).ConclusionsThrough a systematic, rigorous, and participatory process involving international scientists and stakeholders, a new exercise guideline was formulated for cardiometabolic health benefits. A previously published SCI guideline was endorsed for achieving fitness benefits. These guidelines represent an important step toward international harmonization of exercise guidelines for adults with SCI, and a foundation for developing exercise policies and programs for people with SCI around the world.

Raphe-spinal neurons display an age-dependent differential capacity for neurite outgrowth compared to other brainstem-spinal populations

Functional regeneration of brainstem-spinal pathways occurs in the developing chick when the spinal cord is severed prior to embryonic day (E) 13. Functional spinal cord regeneration is not observed in animals injured after E13. This developmental transition from a permissive to a restrictive repair period may be due to the formation of an extrinsic inhibitory environment preventing axonal growth, and/or an intrinsic inability of mature neurons to regenerate. Here, we investigated the capacity of specific populations of brainstem-spinal projection neurons to regrow neurites in vitro from young (E8) versus mature (E17) brainstem explants. A crystal of carbocyanine dye (DiI) was implanted in ovo into the E5 cervical spinal cord to retrogradely label brainstem-spinal projection neurons. Three or 12 days later, discrete regions of the brainstem containing DiI-labeled neurons were dissected to produce explant cultures grown in serum-free media on laminin substrates. The subsequent redistribution of DiI into regenerating processes permitted the study of in vitro neurite outgrowth from identified brainstem-spinal neurons. When explanted on E8, i.e., an age when brainstem-spinal neurons are normally elongating through the spinal cord and are capable of in vivo functional regeneration, robust neurite outgrowth was observed from all brainstem populations, including rubro-, reticulo-, vestibulo-, and raphe-spinal neurons. In contrast, when explanted on E17, robust neurite outgrowth was seen only from raphe-spinal neurons. Neurite outgrowth from raphe-spinal neurons was 5-hydroxy-tryptamine immunoreactive. This study demonstrates that in growth factor-free environments with permissive growth substrates, neurite outgrowth from brainstem-spinal neurons is dependent on both neuronal age and phenotype.

Emerging therapeutic targets in caspase-dependent disease

Caspases are cysteine proteases which serve as the central executioners of apoptotic pathways induced by a wide variety of stimuli. Deregulation of apoptosis and caspase activity contributes to a large number of pathological conditions, including neurodegenerative disorders, stroke, autoimmune disease, disorders involving chronic inflammation, and cancer. Thus, caspases are prime targets for therapeutic interventions aimed at treating these conditions. This review will examine the structure and function of caspases and highlight the findings of applications that inhibit or promote caspase activity in animal models in order to illuminate potential avenues for caspase-regulating drug development.

Transformational mentoring: Leadership behaviors of spinal cord injury peer mentors.

Objective: The purpose of this study was to investigate the leadership behaviors of spinal cord injury (SCI) peer mentors and examine whether behaviors of peer mentors align with the tenets of transformational leadership theory. Method: A total of 12 SCI peer mentors aged 28–75 (M = 49.4) who had between 3 and 56 years (M = 13.9) of mentoring experience were recruited for the study. Utilizing a qualitative methodology (informed by a social constructionist approach), each mentor engaged in a semistructured interview about their experiences as a peer mentor. Interviews were transcribed verbatim and subjected to a directed content analysis. Results: SCI peer mentors reported using mentorship behaviors and engaging with mentees in a manner that closely aligns with the core components of transformational leadership theory: idealized influence, inspirational motivation, individualized consideration, and intellectual stimulation. A new subcomponent of inspirational motivation described as ‘active promotion of achievement’ was also identified and may be unique to the context of peer mentorship. Conclusions: SCI peer mentors inherently use behaviors associated with transformational leadership theory when interacting with mentees. The results from this study have the potential to inform SCI peer mentor training programs about specific leadership behaviors that mentors could be taught to use and could lead to more effective mentoring practices for people with SCI.

Dexamethasone and flavor neophobia in hamsters

The effects of dexamethasone (DEX) on the reactions of male and female hamsters to novel flavors were studied in an attempt to assess the role of novelty-induced stress in flavor neophobia. DEX is a synthetic glucocorticoid of wide-ranging effects including the blocking of adrenal response to stress in pregnant hamsters. If reactions to novel flavors are mediated by stress, DEX-treated hamsters should show an attenuation of flavor neophobia in relation to nontreated animals. Neophobic reactions were assessed by comparing the hamsters’ latency to pouch a novel-flavored nut in comparison with a familiar unflavored one. Flavor neophobia was manifested in response to novel salty, sweet, or sour nuts when male and female hamsters were tested under the control (no-DEX) condition. Similar results were obtained with DEX-treated animals, with one exception. There was an interaction indicating that DEX-treated females showed enhanced neophobic reactions to a sucrose-flavored nut. The theoretical implications of these findings are discussed.