Effect of Acute Administration of Angiopoietin-1 in Experimental Traumatic Spinal Cord Injury: Magnetic Resonance Imaging and Neurobehavioral Studies

Spinal cord injury (SCI) is a devastating condition that affects people in the prime of their lives. A myriad of vascular events occur after SCI, each of which contributes to the evolving pathology. The primary trauma causes mechanical damage to blood vessels, resulting in hemorrhage. The blood-spinal cord barrier (BSCB), a neurovascular unit that limits passage of most agents from systemic circulation to the central nervous system, breaks down, resulting in inflammation, scar formation, and other sequelae. Protracted BSCB disruption may exacerbate cellular injury and hinder neurobehavioral recovery in SCI. In these studies, angiopoietin-1 (Ang1), an agent known to reduce vascular permeability, was hypothesized to attenuate the severity of secondary injuries of SCI. Using longitudinal magnetic resonance imaging (MRI) studies (dynamic contrast-enhanced [DCE]-MRI for quantification of BSCB permeability, highresolution anatomical MRI for calculation of lesion size, and diffusion tensor imaging for assessment of axonal integrity), the acute, subacute, and chronic effects of Ang1 administration after SCI were evaluated. Neurobehavioral assessments were also performed. These non-invasive techniques have applicability to the monitoring of therapies in patients with SCI. In the acute phase of injury, Ang1 was found to reduce BSCB permeability and improve neuromotor recovery. Dynamic contrast-enhanced MRI revealed a persistent compromise of the BSCB up to two months post-injury. In the subacute phase of injury, Ang1’s effect on reducing BSCB permeability was maintained and it was found to transiently reduce axonal integrity. The SCI lesion burden was assessed with an objective method that compared favorably with segmentations from human raters. In the chronic phase of injury, Ang1 resulted in maintained reduction in BSCB permeability, a decrease in lesion size, and improved axonal integrity. Finally, longitudinal correlations among data from the MRI modalities and neurobehavioral assays were evaluated. Locomotor recovery was negatively correlated with lesion size in the Ang1 cohort and positively correlated with diffusion measures in the vehicle cohort. In summary, the results demonstrate a possible role for Ang1 in mitigating the secondary pathologies of SCI during the acute and chronic phases of injury.

Spinal cord injury causes sustained disruption of the blood-testis barrier in the rat.

There is a high incidence of infertility in males following traumatic spinal cord injury (SCI). Quality of semen is frequently poor in these patients, but the pathophysiological mechanism(s) causing this are not known. Blood-testis barrier (BTB) integrity following SCI has not previously been examined. The objective of this study was to characterize the effects of spinal contusion injury on the BTB in the rat. 63 adult, male Sprague Dawley rats received SCI (n = 28), laminectomy only (n = 7) or served as uninjured, age-matched controls (n = 28). Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), BTB permeability to the vascular contrast agent gadopentate dimeglumine (Gd) was assessed at either 72 hours-, or 10 months post-SCI. DCE-MRI data revealed that BTB permeability to Gd was greater than controls at both 72 h and 10 mo post-SCI. Histological evaluation of testis tissue showed increased BTB permeability to immunoglobulin G at both 72 hours- and 10 months post-SCI, compared to age-matched sham-operated and uninjured controls. Tight junctional integrity within the seminiferous epithelium was assessed; at 72 hours post-SCI, decreased expression of the tight junction protein occludin was observed. Presence of inflammation in the testes was also examined. High expression of the proinflammatory cytokine interleukin-1 beta was detected in testis tissue. CD68(+) immune cell infiltrate and mast cells were also detected within the seminiferous epithelium of both acute and chronic SCI groups but not in controls. In addition, extensive germ cell apoptosis was observed at 72 h post-SCI. Based on these results, we conclude that SCI is followed by compromised BTB integrity by as early as 72 hours post-injury in rats and is accompanied by a substantial immune response within the testis. Furthermore, our results indicate that the BTB remains compromised and testis immune cell infiltration persists for months after the initial injury.

Brain fiber tract plasticity in experimental spinal cord injury: diffusion tensor imaging.

Diffusion tensor imaging (DTI) and immunohistochemistry were performed in spinal cord injured rats to understand the basis for activation of multiple regions in the brain observed in functional magnetic resonance imaging (fMRI) studies. The measured fractional anisotropy (FA), a scalar measure of diffusion anisotropy, along the region encompassing corticospinal tracts (CST) indicates significant differences between control and injured groups in the 3 to 4 mm area posterior to bregma that correspond to internal capsule and cerebral peduncle. Additionally, DTI-based tractography in injured animals showed increased number of fibers that extend towards the cortex terminating in the regions that were activated in fMRI. Both the internal capsule and cerebral peduncle demonstrated an increase in GFAP-immunoreactivity compared to control animals. GAP-43 expression also indicates plasticity in the internal capsule. These studies suggest that the previously observed multiple regions of activation in spinal cord injury are, at least in part, due to the formation of new fibers.

Chronic spontaneous activity generated in the somata of primary nociceptors is associated with pain-related behavior after spinal cord injury.

Mechanisms underlying chronic pain that develops after spinal cord injury (SCI) are incompletely understood. Most research on SCI pain mechanisms has focused on neuronal alterations within pain pathways at spinal and supraspinal levels associated with inflammation and glial activation. These events might also impact central processes of primary sensory neurons, triggering in nociceptors a hyperexcitable state and spontaneous activity (SA) that drive behavioral hypersensitivity and pain. SCI can sensitize peripheral fibers of nociceptors and promote peripheral SA, but whether these effects are driven by extrinsic alterations in surrounding tissue or are intrinsic to the nociceptor, and whether similar SA occurs in nociceptors in vivo are unknown. We show that small DRG neurons from rats (Rattus norvegicus) receiving thoracic spinal injury 3 d to 8 months earlier and recorded 1 d after dissociation exhibit an elevated incidence of SA coupled with soma hyperexcitability compared with untreated and sham-treated groups. SA incidence was greatest in lumbar DRG neurons (57%) and least in cervical neurons (28%), and failed to decline over 8 months. Many sampled SA neurons were capsaicin sensitive and/or bound the nociceptive marker, isolectin B4. This intrinsic SA state was correlated with increased behavioral responsiveness to mechanical and thermal stimulation of sites below and above the injury level. Recordings from C- and Aδ-fibers revealed SCI-induced SA generated in or near the somata of the neurons in vivo. SCI promotes the entry of primary nociceptors into a chronic hyperexcitable-SA state that may provide a useful therapeutic target in some forms of persistent pain.

Neuronal and axonal degeneration in experimental spinal cord injury: in vivo proton magnetic resonance spectroscopy and histology.

Longitudinal in vivo proton magnetic resonance spectroscopy (1H-MRS) and immunohistochemistry were performed to investigate the tissue degeneration in traumatically injured rat spinal cord rostral and caudal to the lesion epicenter. On 1H-MRS significant decreases in N-acetyl aspartate (NAA) and total creatine (Cr) levels in the rostral, epicenter, and caudal segments were observed by 14 days, and levels remained depressed up to 56 days post-injury (PI). In contrast, the total choline (Cho) levels increased significantly in all three segments by 14 days PI, but recovered in the epicenter and caudal, but not the rostral region, at 56 days PI. Immunohistochemistry demonstrated neuronal cell death in the gray matter, and reactive astrocytes and axonal degeneration in the dorsal, lateral, and ventral white-matter columns. These results suggest delayed tissue degeneration in regions both rostrally and caudally from the epicenter in the injured spinal cord tissue. A rostral-caudal asymmetry in tissue recovery was seen both on MRI-observed hyperintense lesion volume and the Cho, but not NAA and Cr, levels at 56 days PI. These studies suggest that dynamic metabolic changes take place in regions away from the epicenter in injured spinal cord.

Reduced vascular endothelial growth factor expression in contusive spinal cord injury.

Vascular endothelial growth factor (VEGF) is being investigated as a potential interventional therapy for spinal cord injury (SCI). In the current study, we examined SCI-induced changes in VEGF protein levels using Western blot analysis around the epicenter of injury. Our results indicate a significant decrease in the levels of VEGF(165) and other VEGF isoforms at the lesion epicenter 1 day after injury, which was maintained up to 1 month after injury. We also examined if robust VEGF(165) decrease in injured spinal cords affects neuronal survival, given that a number of reported studies show neuroprotective effect of this VEGF isoform. However, exogenously administered VEGF(165) at the time of injury did not affect the number of sparred neurons. In contrast, exogenous administration of VEGF antibody that inhibits actions of not only VEGF(165) but also of several other VEGF isoforms, significantly decreased number of sparred neurons after SCI. Together these results indicate a general reduction of VEGF isoforms following SCI and that isoforms other than VEGF(165) (e.g., VEGF(121) and/or VEGF(189)) provide neuroprotection, suggesting that VEGF(165) isoform is likely involved in other pathophysiological process after SCI.

Effect of VEGF treatment on the blood-spinal cord barrier permeability in experimental spinal cord injury: dynamic contrast-enhanced magnetic resonance imaging.

Compromised blood-spinal cord barrier (BSCB) is a factor in the outcome following traumatic spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis and vascular permeability. The role of VEGF in SCI is controversial. Relatively little is known about the spatial and temporal changes in the BSCB permeability following administration of VEGF in experimental SCI. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) studies were performed to noninvasively follow spatial and temporal changes in the BSCB permeability following acute administration of VEGF in experimental SCI over a post-injury period of 56 days. The DCE-MRI data was analyzed using a two-compartment pharmacokinetic model. Animals were assessed for open field locomotion using the Basso-Beattie-Bresnahan score. These studies demonstrate that the BSCB permeability was greater at all time points in the VEGF-treated animals compared to saline controls, most significantly in the epicenter region of injury. Although a significant temporal reduction in the BSCB permeability was observed in the VEGF-treated animals, BSCB permeability remained elevated even during the chronic phase. VEGF treatment resulted in earlier improvement in locomotor ability during the chronic phase of SCI. This study suggests a beneficial role of acutely administered VEGF in hastening neurobehavioral recovery after SCI.

Quantitative MRI of spinal cord injury in a rat model: Correlative studies

Serial quantitative and correlative studies of experimental spinal cord injury (SCI) in rats were conducted using three-dimensional magnetic resonance imaging (MRI). Correlative measures included morphological histopathology, neurobehavioral measures of functional deficit, and biochemical assays for N-acetyl-aspartate (NAA), lactate, pyruvate, and ATP. A spinal cord injury device was characterized and provided a reproducible injury severity. Injuries were moderate and consistent to within $\pm$20% (standard deviation). For MRI, a three-dimensional implementation of the single spin-echo FATE (Fast optimum angle, short TE) pulse sequence was used for rapid acquisition, with a 128 x 128 x 32 (x,y,z) matrix size and a 0.21 x 0.21 x 1.5 mm resolution. These serial studies revealed a bimodal characteristic in the evolution in MRI pathology with time. Early and late phases of SCI pathology were clearly visualized in $T\sb2$-weighted MRI, and these corresponded to specific histopathological changes in the spinal cord. Centralized hypointense MRI regions correlated with evidence of hemorrhagic and necrotic tissue, while surrounding hyperintense regions represented edema or myelomalacia. Unexpectedly, $T\sb2$-weighted MRI pathology contrast at 24 hours after injury appeared to subside before peaking at 72 hours after injury. This change is likely attributable to ongoing secondary injury processes, which may alter local $T\sb2$ values or reduce the natural anisotropy of the spinal cord. MRI, functional, and histological measures all indicated that 72 hours after injury was the temporal maximum for quantitative measures of spinal cord pathology. Thereafter, significant improvement was seen only in neurobehavioral scores. Significant correlations were found between quantitated MRI pathology and histopathology. Also, NAA and lactate levels correlated with behavioral measures of the level of function deficit. Asymmetric (rostral/caudal) changes in NAA and lactate due to injury indicate that rostral and caudal segments from the injury site are affected differently by the injury. These studies indicate that volumetric quantitation of MRI pathology from $T\sb2$-weighted images may play an important role in early prediction of neurologic deficit and spinal cord pathology. The loss of $T\sb2$ contrast at 24 hours suggests MR may be able to detect certain delayed mechanisms of secondary injury which are not resolved by histopathology or other radiological modalities. Furthermore, in vivo proton magnetic resonance spectroscopy (MRS) studies of SCI may provide a valuable addition source of information about changes in regional spinal cord lactate and NAA levels, which are indicative of local metabolic and pathological changes. ^

Longitudinal extension of myelomalacia by intramedullary and subdural hemorrhage in a canine model of spinal cord injury

BACKGROUND CONTEXT In canine intervertebral disc (IVD) extrusion, a spontaneous animal model of spinal cord injury, hemorrhage is a consistent finding. In rodent models, hemorrhage might be involved in secondary tissue destruction by biochemical mechanisms. PURPOSE This study aimed to investigate a causal association between the extents of intramedullary, subdural and epidural hemorrhage and the severity of spinal cord damage following IVD extrusion in dogs. STUDY DESIGN/SETTING A retrospective study using histologic spinal cord sections from 83 dogs euthanized following IVD extrusion was carried out. METHODS The degree of hemorrhage (intramedullary, subdural, epidural), the degree of spinal cord damage in the epicenter (white and gray matter), and the longitudinal extent of myelomalacia were graded. Associations between the extent of hemorrhage and the degree of spinal cord damage were evaluated statistically. RESULTS Intramedullary and subdural hemorrhages were significantly associated with the degree of white (p<.001/ p=.004) and gray (both p<.001) matter damage, and with the longitudinal extension of myelomalacia (p<.001/p=.005). Intriguingly, accumulation of hemorrhagic cord debris inside or dorsal to a distended and ruptured central canal in segments distant to the epicenter of the lesion was observed exhibiting a wave-like pattern on longitudinal assessment. The occurrence of this debris accumulation was associated with high degrees of tissue destruction (all p<.001). CONCLUSIONS Tissue liquefaction and increased intramedullary pressure associated with hemorrhage are involved in the progression of spinal cord destruction in a canine model of spinal cord injury and ascending or descending myelomalacia. Functional and dynamic studies are needed to investigate this concept further.

Long-bone fractures in persons with spinal cord injury

STUDY DESIGN Retrospective data analysis. OBJECTIVES To document fracture characteristics, management and related complications in individuals with traumatic spinal cord injury (SCI). SETTING Rehabilitation centre for SCI individuals. METHOD Patients' records were reviewed. Patients with traumatic SCI and extremity fractures that had occurred after SCI were included. Patient characteristics, fractured bone, fracture localisation, severity and management (operative/conservative), and fracture-related complications were extracted. RESULTS A total of 156 long-bone fractures in 107 SCI patients (34 women and 73 men) were identified. The majority of patients were paraplegics (77.6%) and classified as American Spinal Injury Association Impairment Scale A (86.0%). Only the lower extremities were affected, whereby the femur (60.9% of all fractures) was fractured more frequently than the lower leg (39.1%). A total of 70 patients (65.4%) had one fracture, whereas 37 patients (34.6%) had two or more fractures. Simple or extraarticular fractures were most common (75.0%). Overall, 130 (83.3%) fractures were managed operatively. Approximately half of the femur fractures (48.2%) were treated with locking compression plates. In the lower leg, fractures were mainly managed with external fixation (48.8%). Conservative fracture management was applied in 16.7% of the cases and consisted of braces or a well-padded soft cast. Fracture-associated complications were present in 13.5% of the cases but did not differ significantly between operative (13.1%) and conservative (15.4%) fracture management. CONCLUSION SCI was associated with simple or extraarticular fractures of the distal femur and the lower leg. Fractures were mainly managed operatively with a low complication rate.

Description and repeatability of a newly developed spinal cord injury scale for dogs.

The objectives of this study were to describe a new spinal cord injury scale for dogs, evaluate repeatability through determining inter-rater variability of scores, compare these scores to another established system (a modified Frankel scale), and determine if the modified Frankel scale and the newly developed scale were useful as prognostic indicators for return to ambulation. A group of client-owned dogs with spinal cord injury were examined by 2 independent observers who applied the new Texas Spinal Cord Injury Score (TSCIS) and a modified Frankel scale that has been used previously. The newly developed scale was designed to describe gait, postural reactions and nociception in each limb. Weighted kappa statistics were utilized to determine inter-rater variability for the modified Frankel scale and individual components of the TSCIS. Comparisons were made between raters for the overall TSCIS score and between scales using Spearman's rho. An additional group of dogs with surgically treated thoracolumbar disk herniation was enrolled to look at correlation of both scores with spinal cord signal characteristics on magnetic resonance imaging (MRI) and ambulatory outcome at discharge. The actual agreement between raters for the modified Frankel scale was 88%, with a weighted kappa value of 0.93. The TSCIS had weighted kappa scores for gait, proprioceptive positioning and nociception components that ranged from 0.72 to 0.94. Correlation between raters for the overall TSCIS score was Spearman's rho=0.99 (P<0.001). Comparison of the overall TSCIS score to the modified Frankel score resulted in a Spearman's rho value of 0.90 (P<0.001). The modified Frankel score was weakly correlated with the length of hyperintensity of the spinal cord: L2 vertebral body length ratio on mid-sagittal T2-weighted MRI (Spearman's rho=-0.45, P=0.042) as was the overall TSCIS score (Spearman's rho=-0.47, P=0.037). There was also a significant difference in admitting modified Frankel scores (P=0.029) and admitting overall TSCIS scores (P=0.02) between dogs that were ambulatory at discharge and those that were not. Results from this study suggest that the TSCIS is an easy to administer scale for evaluating canine spinal cord injury based on the standard neurological exam and correlates well with a previously described modified Frankel scale.

The use of an electronic von Frey device for evaluation of sensory threshold in neurologically normal dogs and those with acute spinal cord injury

The utility and inter-session repeatability of sensory threshold measurements using an electronic von Frey anesthesiometer (VFA) were assessed in a group of six neurologically normal dogs. Sensory threshold values obtained in neurologically normal dogs were compared to those of dogs with acute spinal cord injury (SCI) caused by intervertebral disc extrusion (n=6) and to a group of neurologically normal dogs with cranial cruciate ligament rupture (CCLR; n=6). Sensory threshold values in neurologically normal dogs were 155.8 ± 37.7 g and 154.7 ± 67.2 g for the left and right pelvic limbs, respectively. The difference in mean sensory threshold values obtained for the group when two distinct testing sessions were compared was not statistically significant (P>0.05). Mean sensory threshold values for the group with SCI were significantly higher than those for neurologically normal dogs at 351.1 ± 116.5 g and 420.3 ± 157.7 g for the left and right pelvic limbs, respectively (P=0.01). A comparison of sensory threshold values for the group with CCLR and neurologically normal dogs was not statistically significant (P>0.05). The modified dorsal technique for VFA described here represents a reliable method to assess sensory threshold in neurologically normal dogs and in those with SCI.

The association of internet use and depression among spinal cord injury population

The purpose of this study was to exam the relationship between internet use and depression among a population of individuals who have sustained spinal cord injury. This was cross-sectional survey design conducted among spinal cord injury (SCI) patients in the Model Spinal Cord Injury System. We included a total of 1,011 SCI-patients who were interviewed face-to-face or by telephone interview over approximately a three year time period (2004–2006). All data were collected through a telephone survey which included the Patient Health Questionnaire-9 (PHQ-9) to assess depression. We examined various scales of this survey, included a reduced 3-item scale (items 1, 2 and 6) to avoid the presence of somatic symptoms among SCI patients from influencing classification of depression. The frequency of internet usage was grouped as daily/weekly/monthly/non user. Covariates examined as possible confounders included demographic characteristics, occupational status, educational level, injury type, daily function of living, pain level, self-perceived health status and satisfaction with life. We observed a negative association between the frequency of internet use and the level of depression. Daily use of internet was associated with lower PHQ-9 score and depression; however this association did not reach statistical significance after for the mentioned covariates. In conclusion, the factors related to lower depression in SCI patients who use the internet are complicated. Daily internet usage was associated with lower levels of depression. The accuracy of 3-item scale needs further validation and investigation. Further study of internet usage pattern in SCI patient is recommended. ^

In vivo longitudinal studies of spinal cord injury and vascular endothelial growth factor treatment

Approximately 12,000 new cases of spinal cord injury (SCI) are added each year to the estimated 259,000 Americans living with SCI. The majority of these patients return to society, their lives forever changed by permanent loss of sensory and motor function. While there are no FDA approved drugs for the treatment of SCI or a universally accepted standard therapy, the current though controversial treatment includes the delivery of high dosages of the corticosteroid methyliprednisolone sodium succinate, surgical interventions to stabilize the spinal column, and physical rehabilitation. It is therefore critically important to fully understand the pathology of injury and determine novel courses and rationally-based therapies for SCI. ^ Vascular endothelial growth factor (VEGF) is an attractive target for treating central nervous system (CNS) injury and disease because it has been shown to influence angiogenesis and neuroprotection. Preliminary studies have indicated that increased vasculature may be associated with functional recovery; therefore exogenous delivery of a pro-angiogenic growth factor such as VEGF may improve neurobehavioral outcome. In addition, VEGF may provide protection from secondary injury and result in increased survival and axonal sprouting. ^ In these studies, SCI rats received acute intraspinal injections of VEGF, the antibody to VEGF, or vehicle control. The effect of these various agents was investigated using longitudinalmulti-modal magnetic resonance imaging (MRI), neuro- and sensory behavioral assays, and end point immunohistochemistry. We found that rats that received VEGF after SCI had increased tissue sparing and improved white matter integrity at the earlier time points as shown by advanced magnetic resonance imaging (MRI) techniques. However, these favorable effects of VEGF were not maintained, suggesting that additional treatments with VEGF at multiple time points may be more beneficial, Histological examinations revealed that VEGF treatment may result in increased oligodendrogenesis and therefore may eventually lead to remyelination and improved functional outcome. ^ On the neurobehavioral studies, treatments with VEGF and Anti-VEGF did not significantly affect performance on tests of open-field locomotion, grid walk, inclined plane, or rearing. However, VEGF treatment resulted in significantly increased incidence of chronic neuropathic pain. This phenomenon could possibly be attributed to the fact that VEGF treatment may promote axonal sprouting and also results in tissue sparing, thereby providing a substrate for the growth of new axons. New connections made by these sprouting axons may involve components of pathways involved in the transmission of pain and therefore result in increased pain in those animals. ^