Radiography used to measure internal spinal cord deformation in an in vivo rat model.

Little is known about the internal mechanics of the in vivo spinal cord during injury. The objective of this study was to develop a method of tracking internal and surface deformation of in vivo rat spinal cord during compression using radiography. Since neural tissue is radio-translucent, radio-opaque markers were injected into the spinal cord. Two tantalum beads (260 µm) were injected into the cord (dorsal and ventral) at C5 of nine anesthetized rats. Four beads were glued to the lateral surface of the cord, caudal and cranial to the injection site. A compression plate was displaced 0.5 mm, 2 mm, and 3 mm into the spinal cord and lateral X-ray images were taken before, during, and after each compression for measuring bead displacements. Potential bead migration was monitored for by comparing displacements of the internal and glued surface beads. Dorsal beads moved significantly more than ventral beads with a range in averages of 0.57-0.71 mm and 0.31-0.35 mm respectively. Bead displacements during 0.5 mm compressions were significantly lower than 2 mm and 3 mm compressions. There was no statistically significant migration of the internal beads. The results indicate the merit of this technique for measuring in vivo spinal cord deformation. The pattern of bead displacements illustrates the complex internal and surface deformations of the spinal cord during transverse compression. This information is needed for validating physical and finite element spinal cord surrogates and to define relationships between loading parameters, internal cord deformation, and biological and functional outcomes.

Activity of the antioxidant enzyme paraoxonase in Argentinean children living at high altitude.

BACKGROUND: Children living at high altitude in San Antonio de los Cobres (SAC), Argentina, were shown to have lower high-density lipoprotein cholesterol (HDL-C) levels than Buenos Aires (BA) children. HDL antioxidant capacity is mainly attributed to paraoxonase1 (PON1). OBJECTIVE: To compare PON1 activity in indigenous SAC vs. BA children. METHODS: A cross-sectional study compared 158 SAC vs. 97 BA children (6-16 years). Anthropometric data and lipoprotein profile were measured. PON1 was evaluated employing paraoxon (PON) and phenylacetate (ARE) activity. RESULTS: The prevalence of overweight/obesity was lower in SAC than in BA children (18.3 vs. 30.9%). Triglycerides (1.34 vs. 0.90 mmol/l), apo B (0.84 vs.0.72 g/l), apo A-I (1.33 vs. 1.27 g/l), and ARE activity (100 vs. 90 µmol/ml/min) were higher, while HDL-C (1.16 vs. 1.32 mmol/l) and PON activity (170 vs. 203 nmol/ml/min) were lower in SAC than in BA. Separate multiple linear regression analyses showed that SAC children had significantly higher triglyceride (Beta -0.38), apo B (Beta -0.34), and ARE (Beta -0.36) plus lower HDL-C (Beta 0.33) and PON (Beta 0.25) compared with BA; adjusted for age, gender, and BMI. CONCLUSION: SAC showed an unfavorable lipoprotein profile, lower PON and higher ARE activities compared with BA children, suggesting the presence of altered HDL metabolism and antioxidant capacity.

Reduced expression of regeneration associated genes in chronically axotomized facial motoneurons.

Chronically axotomized motoneurons progressively fail to regenerate their axons. Since axonal regeneration is associated with the increased expression of tubulin, actin and GAP-43, we examined whether the regenerative failure is due to failure of chronically axotomized motoneurons to express and sustain the expression of these regeneration associated genes (RAGs). Chronically axotomized facial motoneurons were subjected to a second axotomy to mimic the clinical surgical procedure of refreshing the proximal nerve stump prior to nerve repair. Expression of α1-tubulin, actin and GAP-43 was analyzed in axotomized motoneurons using in situ hybridization followed by autoradiography and silver grain quantification. The expression of these RAGs by acutely axotomized motoneurons declined over several months. The chronically injured motoneurons responded to a refreshment axotomy with a re-increase in RAG expression. However, this response to a refreshment axotomy of chronically injured facial motoneurons was less than that seen in acutely axotomized facial motoneurons. These data demonstrate that the neuronal RAG expression can be induced by injury-related signals and does not require acute deprivation of target derived factors. The transient expression is consistent with a transient inflammatory response to the injury. We conclude that transient RAG expression in chronically axotomized motoneurons and the weak response of the chronically axotomized motoneurons to a refreshment axotomy provides a plausible explanation for the progressive decline in regenerative capacity of chronically axotomized motoneurons.

Challenges for defining minimal clinically important difference (MCID) after spinal cord injury.

STUDY DESIGN: This is a review article. OBJECTIVES: This study discusses the following: (1) concepts and constraints for the determination of minimal clinically important difference (MCID), (2) the contrasts between MCID and minimal detectable difference (MDD), (3) MCID within the different domains of International Classification of Functioning, disability and health, (4) the roles of clinical investigators and clinical participants in defining MCID and (5) the implementation of MCID in acute versus chronic spinal cord injury (SCI) studies. METHODS: The methods include narrative reviews of SCI outcomes, a 2-day meeting of the authors and statistical methods of analysis representing MDD. RESULTS: The data from SCI study outcomes are dependent on many elements, including the following: the level and severity of SCI, the heterogeneity within each study cohort, the therapeutic target, the nature of the therapy, any confounding influences or comorbidities, the assessment times relative to the date of injury, the outcome measurement instrument and the clinical end-point threshold used to determine a treatment effect. Even if statistically significant differences can be established, this finding does not guarantee that the experimental therapeutic provides a person living with SCI an improved capacity for functional independence and/or an increased quality of life. The MDD statistical concept describes the smallest real change in the specified outcome, beyond measurement error, and it should not be confused with the minimum threshold for demonstrating a clinical benefit or MCID. Unfortunately, MCID and MDD are not uncomplicated estimations; nevertheless, any MCID should exceed the expected MDD plus any probable spontaneous recovery. CONCLUSION: Estimation of an MCID for SCI remains elusive. In the interim, if the target of a therapeutic is the injured spinal cord, it is most desirable that any improvement in neurological status be correlated with a functional (meaningful) benefit.

Combinatorial treatment of acute spinal cord injury with ghrelin, ibuprofen, C16, and ketogenic diet does not result in improved histologic or functional outcome.

Because of the complex, multifaceted nature of spinal cord injury (SCI), it is widely believed that a combination of approaches will be superior to individual treatments. Therefore, we employed a rat model of cervical SCI to evaluate the combination of four noninvasive treatments that individually have been reported to be effective for acute SCI during clinically relevant therapeutic time windows. These treatments included ghrelin, ibuprofen, C16, and ketogenic diet (KD). These were selected not only because of their previously reported efficacy in SCI models but also for their potentially different mechanisms of action. The administration of ghrelin, ibuprofen, C16, and KD several hours to days postinjury was based on previous observations by others that each treatment had profound effects on the pathophysiology and functional outcome following SCI. Here we showed that, with the exception of a modest improvement in performance on the Montoya staircase test at 8-10 weeks postinjury, the combinatorial treatment with ghrelin, ibuprofen, C16, and KD did not result in any significant improvements in the rearing test, grooming test, or horizontal ladder. Histologic analysis of the spinal cords did not reveal any significant differences in tissue sparing between treatment and control groups. Although single approaches of ghrelin, ibuprofen, C16, and KD have been reported to be beneficial after SCI, our results show that the combination of the four interventions did not confer significant functional or histological improvements in a cervical model of SCI. Possible interactions among the treatments may have negated their beneficial effects, emphasizing the challenges that have to be addressed when considering combinatorial drug therapies for SCI.

Design and biomechanical evaluation of a rodent spinal fixation device.

STUDY DESIGN: An in vitro and in vivo study in rats. OBJECTIVES: To design a novel rat spinal fixation device and investigate its biomechanical effectiveness in stabilizing the spine up to 8 weeks post injury. METHODS: A fixation device made of polyetheretherketone was designed to stabilize the spine via bilateral clamping pieces. The device effectiveness was assessed in a Sprague-Dawley rat model after it was applied to a spine with a fracture-dislocation injury produced at C5-C6. Animals were euthanized either immediately (n=6) or 8 weeks (n=9) post-injury and the C3-T1 segment of the cervical spine was removed for biomechanical evaluation. Segments of intact spinal columns (C3-T1) (n=6) served as uninjured controls. In these tests, anterior-posterior shear forces were applied to the C3 vertebra to produce flexion and extension bending moments at the injury site (peak 12.8 Nmm). The resultant two-dimensional motions at the injury site (that is, C5-C6) were measured using digital imaging and reported as ranges of motion (ROM) or neutral zones (NZ). RESULTS: Flexion/extension ROMs (average±s.d.) were 18.1±3.3°, 19.9±7.5° and 1.5±0.7°, respectively for the intact, injured/fixed, and injured/8-week groups, with the differences being highly significant for the injured/8-week group (P=0.0002). Flexion/extension NZs were 3.4±2.8°, 5.0±2.4°, and 0.7±0.5°, respectively for the intact, injured/fixed, and injured/8-week groups, with the differences being significant for the injured/8-week group (P=0.04). CONCLUSION: The device acutely stabilizes the spine and promotes fusion at the site of injury.

Rehabilitative training and plasticity following spinal cord injury.

Rehabilitative training is currently one of the most successful treatments to promote functional recovery following spinal cord injury. Nevertheless, there are many unanswered questions including the most effective and beneficial design, and the mechanisms underlying the training effects on motor recovery. Furthermore, rehabilitative training will certainly be combined with pharmacological treatments developed to promote the "repair" of the injured spinal cord. Thus, insight into training-induced mechanisms will be of great importance to fine tune such combined treatments. In this review we address current challenges of rehabilitative training and mechanisms involved in promoting motor recovery with the focus on animal models. These challenges suggest that although rehabilitative training appears to be a relatively straight forward treatment approach, more research is needed to optimize its effect on functional outcome in order to enhance our chances of success when combining pharmacological treatments promoting axonal growth and rehabilitative training in the clinic.

Transplantation and repair: combined cell implantation and chondroitinase delivery prevents deterioration of bladder function in rats with complete spinal cord injury.

STUDY DESIGN: Additional examination. In this study, we report changes in bladder function after a combined treatment that was designed to study axonal regeneration after complete spinal cord injury (SCI) in rats. OBJECTIVES: To report effects on bladder function following the administration of a combined treatment for complete SCI. SETTING: University of Alberta, Faculty of Rehabilitation Medicine, Edmonton, Canada. METHODS: Eight rats received Schwann cells in Matrigel-filled guidance channels, olfactory ensheathing glia and chondroitinase ABC at the lesion site following complete thoracic SCI. Controls (n=7) received Matrigel only. Daily bladder examinations were performed. Analysis of bladder size, wall thickness, actin and collagen type III was performed after 14 weeks. RESULTS: Following SCI, both groups regained bladder voiding after 3 weeks. However, 2 weeks later, incontinence was observed in all untreated rats and two treated rats. Post-mortem examination of bladders revealed enlarged bladder sizes. Thicker bladder walls were found in untreated rats, which were composed of disorganized bundles of smooth muscle fibers surrounded by high amounts of collagen (type III). CONCLUSION: We show that the combined treatment prevents collagen deposition in bladder walls and maintains the rat's ability to void efficiently. Although the mechanism responsible for this improvement is unclear, our study shows that the present combinatory therapy can influence bladder function, thus expanding their utility as a broad reparative approach for SCI.

SB203580, a p38 mitogen-activated protein kinase inhibitor, fails to improve functional outcome following a moderate spinal cord injury in rat.

We examined the spatial and temporal expression patterns of active p38 mitogen-activated protein kinase (MAPK), an important regulator of immune cell function, following spinal cord injury (SCI). We further assessed whether administration of SB203580, an inhibitor of p38 MAPK activity, would reduce inflammation, improve tissue sparing, and improve functional outcome after SCI. Adult Wistar rats were subjected to a T9/10 SCI contusion of moderate severity and killed at several time points after injury, whereas sham-injured (control) animals only received a laminectomy. In control animals, active p38 MAPK expression was primarily localized to resting microglia within the spinal cord. Over the first 24 h after SCI, a continuing increase in active p38 MAPK expression was evident in neutrophils and activated microglia (OX42+) surrounding the spinal lesion site. At 15 days post-injury, active p38 MAPK was localized to macrophages (ED1+) that now dominated the lesion site. In addition, active p38 MAPK was localized to macrophages within white matter fiber tracts undergoing degeneration, several segments rostral and caudal to the injury site, which persisted for at least 6 weeks. Overall, our results demonstrate that active p38 MAPK is increased within resident and invading immune cells after SCI contusion injury and, therefore, may be an important target to regulate the inflammatory cascade after SCI. However, intrathecal application of SB203580 failed to improve functional outcome after a moderate SCI contusion.

Class A plexin expression in axotomized rubrospinal and facial motoneurons.

The semaphorin family of guidance molecules plays a role in many aspects of neural development, and more recently semaphorins have been implicated to contribute to the failure of injured CNS neurons to regenerate. While semaphorin expression patterns after neural injury are partially understood, little is known about the expression of their signal transducing transmembrane receptors, the plexins. Therefore, in this study, we compared the expression patterns of all class A plexins (Plxn-A1, A2, A3, A4) in mouse CNS (rubrospinal) and peripheral nervous system (PNS)-projecting (facial) motoneurons for up to two weeks following axonal injury. Using in situ hybridization, immunohistochemistry, and Western blot analysis, in rubrospinal neurons, Plxn-A1 mRNA and protein and Plxn-A4 expression did not change as a result of injury while Plxn-A2 mRNA increased and Plxn-A3 mRNA was undetectable. In facial motoneurons, Plxn-A1, -A3 and -A4 mRNA expression increased, Plxn-A2 mRNA decreased while Plxn-A1 protein expression did not change following injury. We demonstrate that with the exception of the absence of Plxn-A3 mRNA in rubrospinal neurons, both injured rubrospinal (CNS) and facial (PNS) neurons maintain expression of all plexin A family members tested. Hence, there are distinct expression patterns of the individual plexin-A family members suggesting that regenerating rubrospinal and facial motoneurons have a differential ability to transduce semaphorin signals.

Als2-deficient mice exhibit disturbances in endosome trafficking associated with motor behavioral abnormalities.

ALS2 is an autosomal recessive form of spastic paraparesis (motor neuron disease) with juvenile onset and slow progression caused by loss of function of alsin, an activator of Rac1 and Rab5 small GTPases. To establish an animal model of ALS2 and derive insights into the pathogenesis of this illness, we have generated alsin-null mice. Cytosol from brains of Als2(-/-) mice shows marked diminution of Rab5-dependent endosome fusion activity. Furthermore, primary neurons from Als2(-/-) mice show a disturbance in endosomal transport of insulin-like growth factor 1 (IGF1) and BDNF receptors, whereas neuronal viability and endocytosis of transferrin and dextran seem unaltered. There is a significant decrease in the size of cortical motor neurons, and Als2(-/-) mice are mildly hypoactive. Altered trophic receptor trafficking in neurons of Als2(-/-) mice may underlie the histopathological and behavioral changes observed and the pathogenesis of ALS2.

BDNF promotes connections of corticospinal neurons onto spared descending interneurons in spinal cord injured rats.

Although regeneration of injured axons is inhibited within the adult CNS, moderate recovery can be found in patients and animals with incomplete spinal cord injury (SCI). This can be partly attributed to sprouting of spared and injured axons, rostral and caudal to the lesion, respectively. Recently, it has been reported that following a thoracic SCI such sprouting can result in indirect reconnections of the lesioned axons to caudal targets via propriospinal interneurons (PrI). Here, we attempted to further promote this spontaneous repair mechanism by applying the neurotrophic factor BDNF (brain-derived neurotrophic factor), in the vicinity of the cell bodies of lesioned corticospinal neurons or NT-3, intrathecally to the cervical spinal cord. We performed a dorsal over-hemisection at the thoracic spinal cord sparing only the left ventrolateral quadrant. This type of lesion did not promote sprouting of injured corticospinal axons or re-routing via commissural PrI. Also, in rats that received NT-3 at the cervical enlargement, no increase in sprouting was found. However, animals receiving BDNF at the cell bodies of lesioned corticospinal neurons showed a significant increase in collateral sprouting and in the number of contacts with PrI. This was not observed when BDNF was administered to unlesioned animals. Although no statistical difference in the horizontal ladder walking was found between the groups, the increase in collateral sprouting and in the number of contacts correlated with the functional recovery. Hence, cell body treatment can promote plasticity of the injured CNS and may be a valuable treatment approach in conjunction with local regeneration promoting strategies.

Expression and functions of galectin-1 in sensory and motoneurons.

Galectin-1 (Gal1) was the first identified member of the galectin family of beta-galactosidase-binding proteins. Gal1 has important roles in processes fundamental to growth and survival of an organism, including cell adhesion, cell proliferation and apoptosis, and is expressed in many tissues, including the nervous system. In the 1980s, research focused on the developmental regulation of Gal1 expression during neurogenesis. Gal1 was found to be expressed mainly in peripherally-projecting neurons beginning early in neurogenesis, and its expression is maintained at high levels in subpopulations of these neurons in the adult rodent. Although the expression pattern of Gal1 implied that it may be involved in axonal guidance or targeting of subsets of sensory and motoneurons, possible roles of Gal1 in the nervous system had not been confirmed until recently. Gal1 has since been shown to be required for the proper guidance of subsets of primary olfactory axons (to targets in the olfactory bulb) and of primary somatosensory axons (to targets in the superficial dorsal horn). In addition, Gal1 has been implicated in the regenerative response of axons following peripheral nerve injury. Gal1 has been shown to promote axonal regeneration through the activation of macrophages. Also, Gal1 may act within the injured neuron to enhance regrowth: the injury-induced regulation of Gal1 in numerous types of peripherally- and centrally-projecting neurons correlates positively with the regenerative potential of their axons. In this review, we discuss the expression pattern of Gal1 in sensory and motoneurons, and the potential roles of Gal1 in development, axonal regeneration and neuropathic pain.

Regulation of neuronal and glial galectin-1 expression by peripheral and central axotomy of rat primary afferent neurons.

Galectin-1 (Gal1) is an endogenously-expressed protein important for the embryonic development of the full complement of primary sensory neurons and their synaptic connections in the spinal cord. Gal1 also promotes axonal regeneration following peripheral nerve injury, but the regulation of Gal1 by axotomy in primary afferent neurons has not yet been examined. Here, we show by immunohistochemistry and in situ hybridization that Gal1 expression is differentially regulated by peripheral nerve injury and by dorsal rhizotomy. Following peripheral nerve injury, the proportion of Gal1-positive DRG neurons was increased. An increase in the proportion of large-diameter DRG neurons immunopositive for Gal1 was paralleled by an increase in the depth of immunoreactivity in the dorsal horn, where Gal1-positive terminals are normally restricted to laminae I and II. Dorsal rhizotomy did not affect the proportions of neurons containing Gal1 mRNA or protein, but did deplete the ipsilateral dorsal horn of Gal1 immunoreactivity, indicating that it is transported centrally by dorsal root axons. Dorsal rhizotomy also resulted in an increase in Gal1 mRNA the nerve peripheral to the PNS-CNS interface (likely within Schwann cells and/or macrophages), and to a lesser extent within deafferented spinal cord regions undergoing Wallerian degeneration. This latter increase was notable in the dorsal columns and along the prior trajectories of myelinated afferents into the deeper dorsal horn. These results show that neuronal and glial expressions of Gal1 are tightly correlated with regenerative success. Thus, the differential expression pattern of Gal1 following peripheral axotomy and dorsal rhizotomy suggests that endogenous Gal1 may be a factor important to the regenerative response of injured axons.

Altered primary afferent anatomy and reduced thermal sensitivity in mice lacking galectin-1.

The transmission of nociceptive information occurs along non-myelinated, or thinly myelinated, primary afferent axons. These axons are generally classified as peptidergic (CGRP-expressing) or non-peptidergic (IB4-binding), although there is a sub-population that is both CGRP-positive and IB4-binding. During neuronal development and following injury, trophic factors and their respective receptors regulate their survival and repair. Recent reports also show that the carbohydrate-binding protein galectin-1 (Gal1), which is expressed by nociceptive primary afferent neurons during development and into adulthood, is involved in axonal pathfinding and regeneration. Here we characterize anatomical differences in dorsal root ganglia (DRG) of Gal1 homozygous null mutant mice (Gal1(-/-)), as well as behavioural differences in tests of nociception. Gal1(-/-) mice have a significantly reduced proportion of IB4-binding DRG neurons, an increased proportion of NF200-immunoreactive DRG neurons, increased depth of central terminals of IB4-binding and CGRP-immunoreactive axons in the dorsal horn, and a reduced number of Fos-positive second order neurons following thermal (cold or hot) stimulation. While there is no difference in the total number of axons in the dorsal root of Gal1(-/-) mice, there are an increased number of myelinated axons, suggesting that in the absence of Gal1, neurons that are normally destined to become IB4-binding instead become NF200-expressing. In addition, mice lacking Gal1 have a decreased sensitivity to noxious thermal stimuli. We conclude that Gal1 is involved in nociceptive neuronal development and that the lack of this protein results in anatomical and functional deficits in adulthood.

Prolonged local neurotrophin-3 infusion reduces ipsilateral collateral sprouting of spared corticospinal axons in adult rats.

The corticospinal tract is widely used to study regeneration and is essential for voluntary movements in humans. In young rats, corticospinal axons on the uninjured side sprout and grow into the denervated side. Neurotrophin-3 (NT-3) induces such crossed collateral sprouting in adults. We investigated whether local intraspinal NT-3 infusions would promote collateral sprouting of spared corticospinal terminals from within a partially denervated side, as this would be more appropriate for enhancing function of unilateral and specific movements. Adult rats received a partial bilateral transection of the pyramids, leaving approximately 40% of each tract intact. Vehicle or vehicle plus NT-3 (3 or 10 microg/day) was infused for 14 days into the left side of the cervical (C5/6) or lumbar (L2) cord. The corticospinal processes on the left side were anterogradely traced with cholera toxin B (CTB; which labeled gray matter processes more robustly than biotinylated dextran amine) injected into the front or hind limb area of the right sensorimotor cortex, respectively, 3 days before analysis. Unexpectedly, approximately 40% fewer CTB-labeled corticospinal processes were detectable in the cervical or lumbar gray matter of NT-3-treated rats than in vehicle-infused ones. Vehicle-infused injured rats had more corticospinal processes in the center of the cord than normal rats, evidence for lesion-induced collateral sprouting. NT-3 caused sprouting of local calcitonin gene-related peptide-positive fibers. These results suggest that NT-3 reduces collateral sprouting of spared corticospinal axons from within the denervated regions, possibly because of the injury environment or by increasing sprouting of local afferents. They identify an unexpected context-dependent outgrowth inhibitory effect of NT-3.

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.

Galectin-1 expression correlates with the regenerative potential of rubrospinal and spinal motoneurons.

Axotomized spinal motoneurons are able to regenerate to their peripheral targets, whereas injured rubrospinal neurons that lie completely within the CNS fail to regenerate. The differing cell body reactions to axotomy of these two neuronal populations have been implicated in their disparate regenerative ability. Recently, the lectin galectin-1 has been shown to be involved in both spinal motoneurons and primary afferent regeneration. Using in situ hybridization, we compared the endogenous galectin-1 mRNA expression in spinal motoneurons and rubrospinal neurons after axotomy. We found that 7 and 14 days after axotomy, galectin-1 mRNA increased in spinal motoneurons but decreased in rubrospinal neurons. Infusion of the brain-derived neurotrophic factor into the vicinity of the injured rubrospinal nucleus, which we have previously shown to increase the regenerative capacity of rubrospinal neurons, significantly increased galectin-1 mRNA compared with uninjured control levels. Thus, the expression of galectin-1 in neurons correlates with the regenerative propensity.

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

Axotomy 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.