Lowell T. McPhail

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.

The contribution of activated phagocytes and myelin degeneration to axonal retraction/dieback following spinal cord injury

Myelin‐derived molecules inhibit axonal regeneration in the CNS. The Long–Evans Shaker rat is a naturally occurring dysmyelinated mutant, which although able to express the components of myelin lacks functional myelin in adulthood. Given that myelin breakdown exposes axons to molecules that are inhibitory to regeneration, we sought to determine whether injured dorsal column axons in a Shaker rat would exhibit a regenerative response absent in normally myelinated Long–Evans (control) rats. Although Shaker rat axons did not regenerate beyond the lesion, they remained at the caudal end of the crush site. Control rat axons, in contrast, retracted and died back from the edge of the crush. The absence of retraction/dieback in Shaker rats was associated with a reduced phagocytic reaction to dorsal column crush around the caudal edge of the lesion. Systemic injection of minocycline, a tetracycline derivative, in control rats reduced both the macrophage response and axonal retraction/dieback following dorsal column injury. In contrast, increasing macrophage activation by spinal injection of the yeast particulate zymosan had no effect on axonal retraction/dieback in Shaker rats. Schwann cell invasion was reduced in minocycline‐treated control rats compared with untreated control rats, and was almost undetectable in Shaker rats, suggesting that like axonal retraction/dieback, spinal Schwann cell infiltration is dependent upon macrophage‐mediated myelin degeneration. These results indicate that following spinal cord injury the phagocyte‐mediated degeneration of myelin and subsequent exposure of inhibitory molecules to the injured axons contributes to their retraction/dieback.

Axonal reinjury reveals the survival and re-expression of regeneration-associated genes in chronically axotomized adult mouse motoneurons.

Recently, we reported that chronically axotomized rubrospinal neurons survive for up to 1 year in an atrophied state. This finding contrasted previous work suggesting the death of up to 50% of the neurons over time. In the adult mouse, the majority of facial motoneurons appear to be lost as a result of chronic nerve resection. Here, we sought to determine if chronically resected adult mouse facial motoneurons, like rubrospinal neurons, survive in an atrophied state. To test this hypothesis, we asked whether a second nerve injury, 10 weeks after an initial nerve resection, could stimulate a regenerative cell body response. After chronic resection (10 weeks), mouse facial motoneurons underwent atrophy resulting in a loss of countable neuronal cell bodies. In addition, the motoneurons failed to maintain their initial increase in expression of GAP-43 and alpha-tubulin mRNA. Reinjury of 10-week chronically resected facial motoneurons by the removal of the neuroma reversed the atrophy of the cell bodies and increased the percentage of identifiable cell bodies from 36% of contralateral to 79% in C57BL/6-C3H mice and from 28% of contralateral to 40% in Balb/c mice. Moreover, the reinjured motoneurons displayed an increase in GAP-43 and alpha-tubulin mRNA expression. The results of this study indicate that a second axon injury stimulates regenerative cell body responses in chronically resected mouse facial motoneurons and suggest previous studies using this model may have overestimated the number of dying motoneurons.

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.

A soluble Nogo receptor differentially affects plasticity of spinally projecting axons

In the central nervous system, regeneration of injured axons and sprouting of intact axons are suppressed by myelin‐derived molecules that bind to the Nogo receptor (NgR). We used a soluble form of the NgR (sNgR), constructed as an IgG of the human NgR extracellular domain, to manipulate plasticity of uninjured primary afferent and descending monoaminergic projections to the rat spinal cord following dorsal rhizotomy. Rats with quadruple dorsal rhizotomies were treated with intrathecal sNgR or saline, or were left untreated for 2 weeks. Rhizotomy alone resulted in sprouting of serotonergic axons and to a lesser extent, tyrosine‐hydroxylase (TH)‐expressing axons, while axons expressing dopamine‐β‐hydroxylase (DβH) were unaffected. Human IgG immunohistochemistry revealed that sNgR infused into the intrathecal space penetrated approximately 300 µm into spinal white and grey matter. Separate axonal populations differed in their responses to intrathecal sNgR: TH‐expressing and DβH‐expressing axons responded most and least vigorously, respectively. Serotonergic axons were identified by serotonin (5‐HT) or serotonin transporter (SERT) immunohistochemistry. Interestingly, a large increase in 5‐HT compared to SERT‐positive axons density in both saline and sNgR‐treated rats indicated that serotonergic axons both sprouted and increased their transmitter content in response to rhizotomy and sNgR treatment. Calcitonin gene‐related peptide‐positive axons were largely depleted ipsilaterally by rhizotomy, and sNgR increased axon density only in deeper contralateral laminae (III–V). GAP‐43 immunohistochemistry revealed a small increase in axon density following dorsal rhizotomy that was further augmented by sNgR treatment. These results reveal a differential effect of myelin antagonism on distinct populations of spinally projecting axons.

Galectin-1 in regenerating motoneurons.

The exogenous application of recombinant galectin‐1 has recently been shown to promote the rate of peripheral nerve regeneration. Endogenous neuronal galectin‐1 expression has recently been demonstrated to increase after axotomy. Here we demonstrate a significant increase in the endogenous neuronal expression of galectin‐1 mRNA in facial motoneurons after either a nerve resection or crush injury in mice. This increase in galectin‐1 expression was due in part to the loss of target‐derived factor(s) as indicated by both the return of galectin‐1 expression to control levels following target re‐innervation and the increase in galectin‐1 expression after blockade of axonal transport by an interneuronal colchicine injection. Furthermore, interneuronal injections of glial‐derived neurotrophic factor into the uninjured nerve also increased galectin‐1 mRNA expression within facial motoneurons suggesting that positive signals may also be involved in the regulation of galectin‐1 expression. Galectin‐1 null mutant mice showed an attenuated rate of functional recovery of whisking movement after a facial nerve crush.

Endogenous TrkB Ligands Suppress Functional Mechanosensory Plasticity in the Deafferented Spinal Cord

Dorsal root injury (DRI) disrupts the flow of sensory information to the spinal cord. Although primary afferents do not regenerate to their original targets, spontaneous recovery can, by unknown mechanisms, occur after DRI. Here, we show that brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), but not nerve growth factor or neurotrophin-4, are upregulated in the spinal gray matter after DRI. Because endognous BDNF and NT-3 have well established roles in synaptic and axonal plasticity, we hypothesized that they contributed to spontaneous recovery after DRI. We first developed a model of DRI-induced mechanosensory dysfunction: rat C7/8 DRI produced a deficit in low-threshold cutaneous mechanosensation that spontaneously improved within 10 d but did not recover completely. To determine the effects of endogenous BDNF and NT-3, we administered TrkB-Fc or TrkC-Fc fusion proteins throughout the recovery period. To our surprise, TrkB-Fc stimulated complete recovery of mechanosensation by 6 d after DRI. It also stimulated mechanosensory axon sprouting but prevented deafferentation-induced serotonergic sprouting. TrkC-Fc had no effect on low-threshold mechanosensory behavior or axonal plasticity. There was no mechanosensory improvement with single-bolus TrkB-Fc infusions at 10 d after DRI (despite significantly reducing rhizotomy-induced cold pain), indicating that neuromodulatory effects of BDNF did not underlie mechanosensory recovery. Continuous infusion of the pan-neurotrophin antagonist K252a also stimulated behavioral and anatomical plasticity, indicating that these effects of TrkB-Fc treatment occurred independent of signaling by other neurotrophins. These results illustrate a novel, plasticity-suppressing effect of endogenous TrkB ligands on mechanosensation and mechanosensory primary afferent axons after spinal deafferentation.

In vivo application of mitochondrial pore inhibitors blocks the induction of apoptosis in axotomized neonatal facial motoneurons

AbstractAxotomy induces apoptosis in motoneurons of neonatal rodents. To identify the key players in motoneuron apoptosis, we assessed the progression of apoptosis at 4 h intervals following facial motoneuron axotomy. The mitochondrial release of cytochrome c, caspase-3 activation and nuclear condensation were first observed in the motoneuron cell bodies 16 h postaxotomy. In vivo application of inhibitors of the mitochondrial permeability transition pore, Bongkrekic acid and cyclosporin A prevented cytochrome c release as well as caspase-3 activation and attenuated motoneuron apoptosis. Similarly, in vivo application of RU360, an inhibitor of the mitochondrial calcium uniporter, also protected axotomized motoneurons from apoptosis. Taken together, our results show that cytochrome c release and subsequent caspase-3 activation are critical events that precipitate the apoptotic death of axotomized neonatal motoneurons in vivo. In addition, these results provide evidence that application of mitochondrial pore inhibitors in vivo can block the induction of apoptosis following motoneuron axotomy.

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.