Antagonism of purinergic signalling improves recovery from traumatic brain injury.

The recent public awareness of the incidence and possible long-term consequences of traumatic brain injury only heightens the need to develop effective approaches for treating this neurological disease. In this report, we identify a new therapeutic target for traumatic brain injury by studying the role of astrocytes, rather than neurons, after neurotrauma. We use in vivo multiphoton imaging and show that mechanical forces during trauma trigger intercellular calcium waves throughout the astrocytes, and these waves are mediated by purinergic signalling. Subsequent in vitro screening shows that astrocyte signalling through the 'mechanical penumbra' affects the activity of neural circuits distant from the injury epicentre, and a reduction in the intercellular calcium waves within astrocytes restores neural activity after injury. In turn, the targeting of different purinergic receptor populations leads to a reduction in hippocampal cell death in mechanically injured organotypic slice cultures. Finally, the most promising therapeutic candidate from our in vitro screen (MRS 2179, a P2Y1 receptor antagonist) also improves histological and cognitive outcomes in a preclinical model of traumatic brain injury. This work shows the potential of studying astrocyte signalling after trauma to yield new and effective therapeutic targets for treating traumatic brain injury.

A new subject-specific skin correction factor for three-dimensional kinematic analysis of the scapula.

Noninvasive measurement of scapular kinematics using skin surface markers presents technical challenges due to the relative movement between the scapula and the overlying skin. The objectives of this study were to develop a noninvasive subject-specific skin correction factor that would enable a more accurate measurement of scapular kinematics and evaluate this new technique via comparison with a gold standard for scapular movement. Scapular kinematics were directly measured using bone pins instrumented with optoelectronic marker carriers in eight healthy volunteers while skin motion was measured simultaneously with optoelectronic markers attached to the skin surface overlying the scapula. The relative motion between the skin markers and the underlying scapula was estimated over a range of humeral orientations by palpating and digitizing bony landmarks on the scapula and then used to calculate correction factors that were weighted by humeral orientation. The scapular kinematics using these correction factors were compared with the kinematics measured via the bone pins during four arm movements in the volunteers: abduction, forward reaching, hand behind back, and horizontal adduction. The root-mean-square (rms) errors for the kinematics determined from skin markers without the skin correction factors ranged from 5.1 deg to 9.5 deg while the rms errors with the skin correction factors ranged from 1.4 deg to 3.0 deg. This technique appeared to perform well for different movements and could possibly be extended to other applications.

Anterior fracture-dislocation is more severe than lateral: a biomechanical and neuropathological comparison in rat thoracolumbar spine.

ABSTRACT Fracture-dislocation is one of the most common causes of spinal cord injury (SCI) in human adults, yet it is not widely studied experimentally. Clinical studies have found that anterior fracture-dislocation occurs more commonly and produces greater neurological deficit than lateral fracture-dislocation. However, the effect of loading direction on SCI neuropathology has not been investigated experimentally and the reasons behind these clinical differences are not known. Thoracolumbar vertebrae T12-L1 of anaesthetized rats were dislocated anteriorly or laterally by 9 mm at 220 mm/sec. Spinal cord sections from animals euthanized at 1, 3, and 6 h post-injury, were stained with hematoxylin and eosin (H&E) to detect hemorrhage, the pathologic accumulation of beta-amyloid precursor protein (betaAPP) in white matter axons, and degenerating neurons (Fluoro-Jade and loss of NeuN) in the gray matter. The vertebral fracture load and maximum load were similar for both directions of dislocation; however, vertebral fracture occurred at 4.3 mm (+/-1.5 mm SD) during anterior dislocation compared to 1.1 mm (+/-0.7 mm SD) during lateral dislocation (p < 0.001). betaAPP accumulation and reduction of NeuN immunoreactivity (IR) were greatest along a diagonal band across the spinal cord angled at 45 degrees to the direction of loading (in different planes for each loading direction). Hemorrhage volume (p < 0.05), betaAPP-IR, and reduction of NeuN-IR (p < 0.05 in ventral horns) were more pronounced following anterior dislocation. In addition, there was a different spatial distribution of axonal damage for each direction of dislocation. The findings of this study may explain the greater severity of anterior fracture-dislocation observed clinically and reinforces the need to experimentally model differing human SCIs.

Contusion, dislocation, and distraction: primary hemorrhage and membrane permeability in distinct mechanisms of spinal cord injury.

OBJECT: In experimental models of spinal cord injury (SCI) researchers have typically focused on contusion and transection injuries. Clinically, however, other injury mechanisms such as fracture-dislocation and distraction also frequently occur. The objective of the present study was to compare the primary damage in three clinically relevant animal models of SCI. METHODS: Contusion, fracture-dislocation, and flexion-distraction animal models of SCI were developed. To visualize traumatic increases in cellular membrane permeability, fluorescein-dextran was infused into the cerebrospinal fluid prior to injury. High-speed injuries (approaching 100 cm/second) were produced in the cervical spine of deeply anesthetized Sprague-Dawley rats (28 SCI and eight sham treated) with a novel multimechanism SCI test system. The animals were killed immediately thereafter so that the authors could characterize the primary injury in the gray and white matter. Sections stained with H & E showed that contusion and dislocation injuries resulted in similar central damage to the gray matter vasculature whereas no overt hemorrhage was detected following distraction. Contusion resulted in membrane disruption of neuronal somata and axons localized within 1 mm of the lesion epicenter. In contrast, membrane compromise in the dislocation and distraction models was observed to extend rostrally up to 5 mm, particularly in the ventral and lateral white matter tracts. CONCLUSIONS: Given the pivotal nature of hemorrhagic necrosis and plasma membrane compromise in the initiation of downstream SCI pathomechanisms, the aforementioned differences suggest the presence of mechanism-specific injury regions, which may alter future clinical treatment paradigms.

Three-dimensional rotation of the scapula during functional movements: an in vivo study in healthy volunteers.

The goal of this study was to measure 3-dimensional shoulder motion by use of a direct invasive technique during 4 different arm movements in healthy volunteers. Eight subjects with healthy shoulders were recruited. Optoelectronic marker carriers (ie, infrared light-emitting diodes) were mounted on bone pins, which were inserted into the lateral scapular spine. Subjects performed 4 different arm movements while the motion was being recorded by a precision optoelectronic camera. Joint angles were calculated in 3 dimensions. Intraclass correlation coefficients and root-mean-square differences were calculated as measures of reliability. During abduction, the scapula tipped posteriorly (44 degrees +/- 11 degrees), rotated upward (49 degrees +/- 7 degrees), and rotated externally (27 degrees +/- 11 degrees). For reaching, the scapula consistently rotated upward (17 degrees +/- 3 degrees) and rotated internally (18 degrees +/- 6 degrees) whereas tipping was generally less than 10 degrees (5 degrees +/- 2 degrees). Overall, the range of scapular movement for the hand behind the back was small and variable, with most rotations not exceeding 15 degrees. For horizontal adduction, the scapula tipped anteriorly (8 degrees +/- 3 degrees), rotated upward (5 degrees +/- 2 degrees), and rotated internally (27 degrees +/- 6 degrees). These scapular rotations provide normative data that will be useful for diagnosing scapular dysfunction.

The effect of shoulder arthroplasty on humeral strength: an in vitro biomechanical investigation.

BACKGROUND: Periprosthetic humeral fractures are a serious complication of shoulder arthroplasty. While adequate reaming of the canal and insertion of an oversized implant optimizes fit, such maneuvers also weaken the bone and predispose it to fracture. METHODS: The impact of the humeral arthroplasty was assessed in vitro on human cadaveric specimens. Strain gauges were attached to the distal diaphyses and the specimens were mounted in a torsion-loading fixture throughout the tests. An initial series examined the effect of reaming of the canal to its clinically appropriate diameter using uniaxial strain gauges. A second series utilized strain rosettes to evaluate the cumulative effects of reaming, broaching, and implant insertion. FINDINGS: Reaming of the canal to its clinically appropriate diameter significantly increased (P=0.007) uniaxial strain measurements by a mean of 30% with five of eight specimens showing increases of over 49% on at least one of four diaphyseal locations. In the second series, the surface strain was significantly affected by arthroplasty (P<0.008). Post-hoc analysis showed that the maximum in-plane shear strain following implant insertion was significantly increased relative to strain levels following reaming and broaching (P<0.009). The direction of the principal strain axes did not significantly change (P>0.46). Unexpected decreases in some strain measurements were observed as the arthroplasty procedure progressed perhaps reflecting overt mechanical failure within the humeral shaft. INTERPRETATION: The strain increase following reaming suggests a reduction in torsional strength by over 33% which is further reduced following broaching and implant insertion. For the practicing surgeon, post-operative strength can be adversely affected by both canal preparation and implant insertion.

Improved RSA accuracy with DLT and balanced calibration marker distributions with an assessment of initial-calibration.

Roentgen stereophotogrammetric analysis (RSA) has been used for over 25 years for accurate micromotion measurement in a wide variety of orthopaedic applications. This study investigated two possible improvements to the method. First, direct linear transformation (DLT) was compared to the traditional RSA reconstruction algorithm. The two methods were considered with respect to standard extrapolation and interpolation calibration cages. Matlab simulations showed that reconstruction accuracy was greatly improved (>60%) by combining DLT with an even distribution of enclosing calibration markers. Second, a benchtop study using phantoms translated at 0.0254-mm intervals showed initial-calibration, followed by removal of the interpolation cage for subsequent exposures, was potentially twice as accurate as self-calibration with an extrapolation cage. These results showed optimizations for the application of RSA when unobstructed space is required.

Histological effects of residual compression sustained for 60 minutes at different depths in a novel rat spinal cord injury contusion model.

Spinal cord injuries are frequently accompanied by persisting residual compression of the spinal cord; however, it remains controversial as to what effect the sustained compression has on neurological damage. The objective of this study was to determine the influence of post-traumatic residual spinal cord compression on the extent and progression of tissue damage within a dynamic thoracic contusion rat model. Twenty-nine male Wistar rats were distributed into one of four groups: spinal cord contusion only, contusion with 40% residual compression, contusion with 90% residual compression, and a surgical control group. A moderate injury was performed (1 mm, 700 mm/sec) with our custom University of British Columbia (UBC) multimechanism apparatus, and the residual compression groups had the impactor tip maintained at 40% or 90% of the initial impact depth (1.0 mm) for 60 min post-injury. All animals were killed at 3 h post-injury, when the spinal cord was harvested and stained for hemorrhage, neuronal damage in the gray matter, and axonal disruption in the white matter. The initial contusion injury immediately damaged tissue beneath the impactor as evidenced by rapid relaxation of the reaction force on the spinal cord during the subsequent compression. Importantly, the rostral-caudal extent of intramedullary hemorrhage was 66% larger after 90% residual spinal cord compression compared to the 40% group (p=0.016). Similarly, the extent of neuronal nuclei lost in different gray matter regions was 60-86% greater after 90% residual compression compared with 40% (p<0.001). Thus, a high level of residual compression of the spinal cord following a moderate contusion injury has the potential to adversely increase the extent of tissue damage, whereas a lower level of residual compression may have little to no effect.

Maximum principal strain correlates with spinal cord tissue damage in contusion and dislocation injuries in the rat cervical spine.

The heterogeneity of the primary mechanical mechanism of spinal cord injury (SCI) is not currently used to tailor treatment strategies because the effects of these distinct patterns of acute mechanical damage on long-term neuropathology have not been fully investigated. A computational model of SCI enables the dynamic analysis of mechanical forces and deformations within the spinal cord tissue that would otherwise not be visible from histological tissue sections. We created a dynamic, three-dimensional finite element (FE) model of the rat cervical spine and simulated contusion and dislocation SCI mechanisms. We investigated the relationship between maximum principal strain and tissue damage, and compared primary injury patterns between mechanisms. The model incorporated the spinal cord white and gray matter, the dura mater, cerebrospinal fluid, spinal ligaments, intervertebral discs, a rigid indenter and vertebrae, and failure criteria for ligaments and vertebral endplates. High-speed (∼ 1 m/sec) contusion and dislocation injuries were simulated between vertebral levels C3 and C6 to match previous animal experiments, and average peak maximum principal strains were calculated for several regions at the injury epicenter and at 1-mm intervals from +5 mm rostral to -5 mm caudal to the lesion. Average peak principal strains were compared to tissue damage measured previously in the same regions via axonal permeability to 10-kD fluorescein-dextran. Linear regression of tissue damage against peak maximum principal strain for pooled data within all white matter regions yielded similar and significant (p<0.0001) correlations for both contusion (R(2)=0.86) and dislocation (R(2)=0.52). The model enhances our understanding of the differences in injury patterns between SCI mechanisms, and provides further evidence for the link between principal strain and tissue damage.

NR2A and NR2B subunits differentially mediate MAP kinase signaling and mitochondrial morphology following excitotoxic insult.

NMDA receptors are essential for neurotransmission and key mediators of synaptic signaling, but they can also trigger deleterious degenerative processes that lead to cell death. Growing evidence suggests that selective blockade of the heterogeneous subunits that comprise the NMDA receptor may enable better control of pharmacotherapies for treating neurological diseases and injuries. We investigated the relationship between NMDAR activation, MAPK signaling, and mitochondrial shape following an excitotoxic insult. NR2A- and NR2B-containing NMDARs differentially mediated acute changes in cytosolic calcium, alterations in mitochondrial morphology, and phosphorylation of the MAPKs ERK and JNK. Activation of NR2A-containing NMDARs was associated with JNK phosphorylation that was neuroprotective in neuronal cultures subjected to excitotoxicity. In contrast, activation of NR2B-containing NMDARs triggered calcium accumulation in mitochondria that was strongly associated with mitochondrial swelling and neuronal cell death. Indeed, while blockade of NR2B-containing receptors was neuroprotective, this protection was lost when NR2A-initiated JNK phosphorylation was inhibited. Given the modest selectivity of the NR2A inhibitor, NVP-AAM077, the results highlight the significance of the relative, rather than absolute, activation of these two NMDA subtypes in modulating cell death pathways. Therefore, the balance between concurrent activation of NR2B-containing and NR2A-containing NMDARs dictates neuronal fate following excitotoxicity.

The placement of skin surface markers for non-invasive measurement of scapular kinematics affects accuracy and reliability.

Abnormal scapular movement is widely believed to be an important factor in clinical pathology of the shoulder joint complex. Validated non-invasive techniques for measuring scapular movement have been developed, but the effect of marker placement on accuracy is unknown. The objective of this study was to determine the accuracy and reliability of different groupings of markers to achieve the best accuracy and reliability for measuring scapular kinematics. Eight healthy young adult subjects were recruited. An optoelectronic marker grid was applied to the skin overlying the scapula. Two bone pins with optoelectronic marker carriers were inserted into the scapula. The accuracy of six surface marker configurations was determined by comparing the measured kinematics with scapular bone pins (the gold standard). Four humeral movements were tested: glenohumeral abduction, glenohumeral horizontal adduction, hand behind back, and forward reaching. All three rotations had a significant difference in the accuracy of the patches (p = 0.04 to p < 0.0001). For posterior tipping there was a significant effect of movement (p = 0.003) and a significant interaction (p < 0.0001). There was also a significant interaction for external rotation (p = 0.001). The marker grouping with the largest cranio-caudal spread had the highest accuracy for measuring posterior tilting (RMS 1.9°). Markers closer to the scapular spine were more accurate for tracking external rotation (RMS 2.0°) while an intermediate grouping of markers were most accurate for quantifying upward rotation (RMS 1.9°). The reliability between days ranged between 3.8° and 7.5° (based on RMS difference between trials) and there was a significant interaction between patch and movement (p < 0.0001). Intraclass correlation coefficients show moderate to good agreement for most arm movements and scapular rotations. Thus, there exists distinct optimal configurations of non-invasive marker locations for accurately measuring scapular kinematics.

The distribution of tissue damage in the spinal cord is influenced by the contusion velocity.

STUDY DESIGN: A rat model of thoracic spinal cord contusion was used to examine the effect of velocity on the primary injury. OBJECTIVES: The overall objective of this study was to determine the effect of the contusion velocity (slow vs. fast) on damage to the spinal cord immediately following mechanical injury. Secondary objectives were to demarcate between damage in the gray and white matters and to observe damage to the mechanical elements of the neurons (i.e., neurofilaments). SUMMARY OF BACKGROUND DATA: Although studies have explored the effect of impact velocity on spinal cord damage and functional deficits, no study has addressed regional tissue damage of the primary injury (e.g., between the gray and white matter) as a function of velocity. METHODS: A modified Spinal Cord Injury Research System generated 1 mm contusions in 24 male, Sprague-Dawley rats (210-320 g) at T10, using slow (3 mm/s) and fast (300 mm/s) velocities. The primary lesion (<2 minutes postinjury) was assessed using hematoxylin and eosin staining for hemorrhage volume and immunostaining for nonphosphorylated heavy neurofilament damage. RESULTS: The volume of hemorrhage in the white matter was significantly increased following fast impact (fast = 0.61 mm3, slow = 0.24 mm3, P = 0.013) whereas the total hemorrhage volume (fast = 1.51 mm, slow = 1.21 mm, P = 0.22) showed no effect. Complete axonal disruption was evident in the fast injury group around the injury epicenter. A significant increase in nonphosphorylated neurofilament staining (P = 0.013) was observed for fast impacts. Hemorrhage in the gray matter was similar between the slow and fast groups, but an increase in neurofilament dephosphorylation was observed in the gray matter following fast contusion (P = 0.03). CONCLUSION: We conclude that contusion velocity has an effect on the magnitude of injury within the white matter during spinal cord injury and the amount of neuronal damage in the gray matter. The results of this study demonstrate the importance of including high impact velocity as a variable in models of spinal cord injury.

Secondary pathology following contusion, dislocation, and distraction spinal cord injuries.

Preclinical studies for spinal cord injury (SCI) have utilized transection and contusion injury paradigms even though human SCIs occur by a spectrum of primary injury mechanisms such as spinal cord contusion from vertebral burst fracture, shearing from fracture-dislocation, and stretching from distraction injuries. We contrasted the neuropathology in animal models mimicking these clinically relevant injuries at an early 3-hour time-point in order to relate patterns of secondary pathology to the primary injury mechanism. Axolemma compromise, detected by the intracellular penetration of dextran-conjugated fluorophores, was localized to the contusion epicentre but extended rostrally following dislocation and distraction injuries. Dextran infused post-trauma revealed extensive axolemma resealing whereas only modest membrane recovery was detected in neuronal somata. Fracture-dislocations produced greater axonal degeneration than either contusion or distraction injuries as evidenced by reduced neurofilament immunostaining in ventral tracts, increased beta-amyloid precursor protein accumulation in lateral funiculi, and a longer lesion in the dorsal corticospinal tract. In the gray matter, cytochrome c release from neuronal mitochondria, indicative of early apoptosis, was detected within the penumbrae of the contusion and dislocation injuries only. Neurons positive for the oxidative stress marker 3-nitrotyrosine were most numerous rostral to the dislocation epicentre. Microglial activation was localized to the contusion epicentre, extended rostro-caudally following dislocation, but was similar to surgical controls after distraction injuries. Reactive astrocytes extended rostro-caudally only following dislocation injuries. Hence, the primary injury mechanism alters the pattern of secondary degeneration indicating that different neuroprotective strategies may ultimately be required for treating distinct clinically relevant SCIs.