Lyn B. Jakeman

Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats

Contusive spinal cord injury (SCI) produces large fluid-, debris- and inflammatory cell-filled cystic cavities that lack structure to support significant axonal regeneration. The recent discovery of stem cells capable of generating central nervous system (CNS) tissues, coupled with success in neurotransplantation strategies, has renewed hope that repair and recovery from CNS trauma is possible. Based on results from several studies using bone marrow stromal cells (MSCs) to promote CNS repair, we transplanted MSCs into the rat SCI lesion cavity to further investigate their effects on functional recovery, lesion morphology, and axonal growth. We found that transplanted MSCs induced hindlimb airstepping--a spontaneous locomotor movement associated with activation of the stepping control circuitry--but did not alter the time course or extent of overground locomotor recovery. Using stereological techniques to describe spinal cord anatomy, we show that MSC transplants occupied the lesion cavity and were associated with preservation of host tissue and white matter (myelin), demonstrating that these cells exert neuroprotective effects. The tissue matrix formed by MSC grafts supported greater axonal growth than that found in specimens without grafts. Moreover, uniform random sampling of axon profiles revealed that the majority of neurites in MSC grafts were oriented with their long axis parallel to that of the spinal cord, suggesting longitudinally directed growth. Together, these studies support further investigation of marrow stromal cells as a potential SCI repair strategy.

Brain-Derived Neurotrophic Factor Stimulates Hindlimb Stepping and Sprouting of Cholinergic Fibers after Spinal Cord Injury

Neurotrophic factors have been proposed as a therapeutic treatment for traumatic brain and spinal cord injury. The present study determined whether exogenous administration of one such factor, brain-derived neurotrophic factor (BDNF), could effect behavioral recovery and/or histopathological changes after spinal cord injury. Adult rats received a mild or moderate contusion injury or complete transection of the mid-thoracic spinal cord. Immediately thereafter, they were infused intrathecally with vehicle or BDNF for 28 days. Behavioral recovery was evaluated for 6 weeks after injury, at which time the rats were sacrificed and the spinal cord tissue was examined histologically. The infusion of BDNF resulted in acute stimulation of hindlimb activity. These effects included activation of alternating airstepping in injured rats when the hindlimbs were unloaded as well as slight improvements in the rate of recovery in open field locomotion score. BDNF infusion was also associated with enhanced growth of cholinergic fibers at the injury epicenter, but did not affect white matter sparing or density of serotonergic axons at or below the injury site. Based on immunohistochemical detection of BDNF protein distribution, these described effects are likely to be mediated by the activation of cells and axons within the central injury region and the along the peripheral rim of the spinal cord. Together, these findings demonstrate that the exogenous infusion of BDNF after spinal trauma can influence postinjury outcome through mechanisms that include acute stimulation of hindlimb activity and neuritogenesis at the injury site.

Behavioral and Histological Outcomes Following Graded Spinal Cord Contusion Injury in the C57Bl/6 Mouse

A computer-controlled electromagnetic spinal cord injury device (ESCID) has been adapted to develop a mouse model of spinal cord contusion injury. In the present study, we have extended this model in C57Bl/6 mice with behavioral and histopathological outcome assessment. Three groups of mice received a laminectomy at the T(9) vertebral level followed by a contusion injury from a predetermined starting load of 1500 dynes. Contusion was produced by rapid displacement of the spinal cord to a peak distance of 0.3, 0.5, or 0.8 mm, with the entire injury and retraction procedure completed over a 23-ms epoch. Control groups received laminectomy alone or complete transection. Functional recovery was examined for 9 weeks after injury using the BBB locomotor rating scale, grid walking, and footprint analysis. Distinct patterns of locomotor recovery were evident across the five groups. Measurements of spared white matter at the epicenter, lesion length, and cross-sectional area of fibronectin-immunopositive scar tissue were also significantly different between injury groups. The severity of injury corresponded with the biomechanical measures recorded at the time of impact as well as with behavioral and histological parameters. The results demonstrate that graded contusion injuries can be produced reliably in mice using the ESCID. The data provide a thorough and quantitative analysis of the effects of contusion injury on long-term behavioral and histological outcome measures in this strain and species.

Operant Conditioning of H-Reflex Can Correct a Locomotor Abnormality after Spinal Cord Injury in Rats

This study asked whether operant conditioning of the H-reflex can modify locomotion in spinal cord-injured rats. Midthoracic transection of the right lateral column of the spinal cord produced a persistent asymmetry in the muscle activity underlying treadmill locomotion. The rats were then either exposed or not exposed to an H-reflex up-conditioning protocol that greatly increased right soleus motoneuron response to primary afferent input, and locomotion was reevaluated. H-reflex up-conditioning increased the right soleus burst and corrected the locomotor asymmetry. In contrast, the locomotor asymmetry persisted in the control rats. These results suggest that appropriately selected reflex conditioning protocols might improve function in people with partial spinal cord injuries. Such protocols might be especially useful when significant regeneration becomes possible and precise methods for reeducating the regenerated spinal cord neurons and synapses are needed for restoring effective function.

Monocyte recruitment and myelin removal are delayed following spinal cord injury in mice with CCR2 chemokine receptor deletion.

The inflammatory response initiated after spinal cord injury (SCI) is characterized by the accumulation of macrophages at the impact site. Monocyte chemoattractant protein‐1 (MCP‐1) is a strong candidate for mediating chemotaxis of monocytes to the injured nervous system. To help in defining the role of MCP‐1 in inflammation after SCI, we evaluated the time course of macrophage accumulation for 2 weeks following a midthoracic spinal cord contusion injury in mice lacking CCR2, a principal receptor for MCP‐1. Mice with a deletion of CCR2 resulted in significantly reduced Mac‐1 immunoreactivity restricted to the lesion epicenter at 7 days postinjury. The regions devoid of Mac‐1 immunoreactivity corresponded to areas of reduced myelin degradation at this time. By 14 days postinjury, however, there were no differences in Mac‐1 staining between CCR2 (+/+) and CCR2 (–/–) mice. Analyses of mRNA levels by RNase protection assay (RPA) revealed increases in MCP‐1 as well as MCP‐3 and MIP‐2 mRNA at 1 day postinjury compared with 7 day postinjury. There were no differences in chemokine expression between CCR2‐deficient mice and wild‐type littermate controls. The CCR2‐deficient mice also exhibited reduced expression of mRNA for chemokine receptors CCR1 and CCR5. Together, these results indicate that chemokines acting through CCR2 contribute to the early recruitment of monocytes to the lesion epicenter following SCI.

Peroxynitrite production and activation of poly (adenosine diphosphate-ribose) synthetase in spinal cord injury.

Peroxynitrite formation and the subsequent activation of the nuclear enzyme, poly (adenosine diphosphate [ADP]‐ribose) synthetase (PARS), has been implicated in the pathogenesis of several neurodegenerative disorders. Here, we demonstrate that nitrotyrosine, an indicator of peroxynitrite generation, and poly (ADP) ribose, a marker of PARS activation, were selectively localized within tissues from spinal cord–injured rats. Our data implicate a role for peroxynitrite production and PARS activation in the development of spinal cord trauma. Ann Neurol 1999;45:120–124

Pegylated Brain-Derived Neurotrophic Factor Shows Improved Distribution into the Spinal Cord and Stimulates Locomotor Activity and Morphological Changes after Injury

The neurotrophin brain-derived neurotrophic factor (BDNF) shows promise for the treatment of central nervous system (CNS) trauma and disease. Effective delivery methods are required, however, for BDNF to be useful as a therapeutic agent. To this end, we examined the penetration of intrathecally infused N-terminal pegylated BDNF (peg-BDNF) compared to similar infusion of native BDNF after spinal cord injury (SCI). Pegylation dramatically improved delivery of BDNF to the spinal cord and induced the expression of Fos in spinal cord neurons. To test whether enhanced delivery would improve the modest effects on behavioral recovery and axonal outgrowth observed with native BDNF infusion, we assessed the efficacy of 2-week 25 microg/day peg-BDNF treatment, beginning 12-24 h (early) or 15 days (delayed) after midthoracic spinal contusion. Similar to native BDNF, early treatment with peg-BDNF accelerated the recovery of stepping in the open-field and acutely stimulated locomotor central pattern generator activity, as seen by the activation of hindlimb airstepping during either period of administration. The infusion of peg-BDNF, regardless of the timing of delivery, was related to enhanced sprouting of putative cholinergic fibers, like that observed after high dose native BDNF treatment. Despite improved delivery, however, neither axonal responses nor the extent of locomotor recovery were enhanced compared to native BDNF treatment. This suggests that alternative strategies, such as neurotrophin treatment in conjunction with cell transplantation techniques, or treatment nearer the cell bodies of target neurons might be employed in an attempt to effect significant repair after SCI.

The Interaction of a New Motor Skill and an Old One: H-Reflex Conditioning and Locomotion in Rats

New and old motor skills can interfere with each other or interact in other ways. Because each skill entails a distributed pattern of activity-dependent plasticity, investigation of their interactions is facilitated by simple models. In a well characterized model of simple learning, rats and monkeys gradually change the size of the H-reflex, the electrical analog of the spinal stretch reflex. This study evaluates in normal rats the interactions of this new skill of H-reflex conditioning with the old well established skill of overground locomotion. In rats in which the soleus H-reflex elicited in the conditioning protocol (i.e., the conditioning H-reflex) had been decreased by down-conditioning, the H-reflexes elicited during the stance and swing phases of locomotion (i.e., the locomotor H-reflexes) were also smaller. Similarly, in rats in which the conditioning H-reflex had been increased by up-conditioning, the locomotor H-reflexes were also larger. Soleus H-reflex conditioning did not affect the duration, length, or right/left symmetry of the step cycle. However, the conditioned change in the stance H-reflex was positively correlated with change in the amplitude of the soleus locomotor burst, and the correlation was consistent with current estimates of the contribution of primary afferent input to the burst. Although H-reflex conditioning and locomotion did not interfere with each other, H-reflex conditioning did affect how locomotion was produced: it changed soleus burst amplitude and may have induced compensatory changes in the activity of other muscles. These results illustrate and clarify the subtlety and complexity of skill interactions. They also suggest that H-reflex conditioning might be used to improve the abnormal locomotion produced by spinal cord injury or other disorders of supraspinal control.

Regional heterogeneity in astrocyte responses following contusive spinal cord injury in mice.

Astrocytes and their precursors respond to spinal cord injury (SCI) by proliferating, migrating, and altering phenotype. This contributes to glial scar formation at the lesion border and gliosis in spared gray and white matter. The present study was undertaken to evaluate astrocyte changes over time and determine when and where interventions might be targeted to alter the astrocyte response. Bromodeoxyuridine (BrdU) was administered to mice 3 days after SCI, and cells expressing BrdU and the astrocyte marker, glial fibrillary acidic protein (GFAP), were counted at 3, 7, and 49 days post‐injury (DPI). BrdU‐labeled cells accumulated at the lesion border by 7 DPI and approximately half of these expressed GFAP. In spared white matter, the total number of BrdU+ cells decreased, while the percentage of BrdU+ cells expressing GFAP increased at 49 DPI. Phenotypic changes were examined using the progenitor marker nestin, the radial glial marker, brain lipid binding protein (BLBP), and GFAP. Nestin was upregulated by 3 DPI and declined between 7 and 49 DPI in all regions, and GFAP increased and remained above naïve levels at all timepoints. BLBP increased early and remained high along the lesion border and spared white matter, but was expressed transiently by cells lining the central canal and in a unique population of small cells found within the lesion and in gray matter rostral and caudal to the border. The results demonstrate that the astrocyte response to SCI is regionally heterogeneous, and suggests astrocyte populations that could be targeted by interventions. J. Comp. Neurol. 518:1370–1390, 2010.

Enhanced axonal growth into a spinal cord contusion injury site in a strain of mouse (129X1/SvJ) with a diminished inflammatory response

After injury in the adult central nervous system, invading and intrinsic cells contribute to the formation of a lesion site that is refractory to axonal growth. To test the hypothesis that the inflammatory response to trauma dictates the extent of axonal growth after spinal cord injury, the time course of lesion evolution was compared in two mouse strains with contrasting cellular responses to peripheral inflammatory challenge. Adult C57Bl/6 and 129X1/SvJ mice received identical contusion injuries to the mid‐thoracic spinal cord and were allowed to recover for 6 hours to 9 weeks. Both strains responded with a rapid, transient increase in chemokine expression, but the magnitude of this early response was slightly reduced in the 129X1/SvJ mice. Morphological indicators of inflammation were similar during the first week postinjury. After 7 days postinjury, however, the cellular responses differed between strains. The C57Bl/6 lesion core was chronically occupied by macrophages, devoid of astrocytes, and contained few axonal profiles. In contrast, as the macrophage density decreased a network of astrocytic processes and axons of central and peripheral origin invaded the center of the lesion site in 129X1Sv/J mice. Growth of axons in the 129X1Sv/J mice was accompanied by increased extravascular laminin in the lesion core and a reduced expression of chondroitin sulfate proteoglycan glycosaminoglycan sidechains in the periphery of the lesion. These results demonstrate that the diminished chronic inflammatory response in 129X1/SvJ mice is associated with enhanced cellular repair and increased axonal growth after spinal cord injury. J. Comp. Neurol. 474:469–486, 2004.