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Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
The role of hemorrhage following spinal-cord injury$ Q1
Patrick Loseya,b,n, Christopher Youngc,1, Emily Krimholtza,1, Re´gis Bordetb,2, Daniel C. Anthonya,b,1 a
Experimental Neuropathology, Department of Pharmacology, University of Oxford, Oxford, UK EA 1046, Pharmacology, Faculty of Medicine, Research Branch, IMPRT, University of Lille North of France, Lille, France c Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK b
art i cle i nfo Article history: Accepted 23 April 2014
ab st rac t Q2 Spinal-cord injury is characterized by primary damage as a direct consequence of mechanical insult, and secondary damage that is partly due to the acute inflammatory response. The extent of any hemorrhage within the injured cord is also known to be
Keywords:
associated with the formation of intraparenchymal cavities and has been anecdotally
Spinal-cord injury
linked to secondary damage. This study was designed to examine the contribution of blood
Hemorrhage
components to the outcome of spinal-cord injury. We stereotaxically microinjected
Inflammation
collagenase, which causes localized bleeding, into the spinal cord to model the hemor-
Models of injury
rhage associated with spinal cord injury in the absence of significant mechanical trauma. Tissue damage was observed at the collagenase injection site over time, and was associated with localized disruption of the blood-spinal-cord barrier, neuronal cell death, and the recruitment of leukocytes. The magnitude of the bleed was related to neutrophil mobilization. Interestingly, the collagenase-induced injury also provoked extended axonal damage. With this model, the down-stream effects of hemorrhage are easily discernible, and the impact of treatment strategies for spinal-cord injury on hemorrhage-related injury can be evaluated. & 2014 Published by Elsevier B.V.
1.
Introduction
Traumatic spinal-cord injury (SCI) causes irreversible axonal damage and neuronal death, resulting in permanent disability. In addition to the initial mechanical injury, a cascade of events
takes place that precipitates further axonal damage and neuronal death long after the primary insult. This cascade of events is collectively termed secondary injury (Sekhon and Fehlings, 2001), and was first postulated as early as 1914 by Allen who suggested that noxious agents present in the
☆
Sources of support: This work was funded by the Medical Research Council under Grant number MRC G0300456. Corresponding author at: Experimental Neuropathology, Department of Pharmacology, University of Oxford, Oxford, UK. Fax: þ44 1865 271853. E-mail addresses: [email protected] (P. Losey), [email protected] (C. Young), [email protected] (E. Krimholtz), [email protected] (R. Bordet), [email protected] (D.C. Anthony). 1 Fax: þ44 1865 271853. 2 Fax: þ33 320446863. n
http://dx.doi.org/10.1016/j.brainres.2014.04.033 0006-8993/& 2014 Published by Elsevier B.V.
Please cite this article as: Losey, P., et al., The role of hemorrhage following spinal-cord injury. Brain Research (2014), http: //dx.doi.org/10.1016/j.brainres.2014.04.033
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hemorrhagic material might cause further damage to the spinal cord (Allen, 1914). The mechanisms of secondary injury include hemorrhage, tissue ischemia, blood-spinal-cordbarrier (BSCB) breakdown, inflammation, and glutamate toxicity, as well as demyelination and apoptotic signaling (Fleming et al., 2006). SCI is associated with mechanical insult to the microvasculature, leading to a primary hemorrhage into the spinal cord. Subsequent to the initial bleeding, there is a formation of secondary petechial hemorrhages in the tissue surrounding the primary lesion that have been shown to be related to a dramatic increase in the extent of the lesion (Gerzanich et al., 2009). Blood infiltrating the central nervous system (CNS) tissue has been shown to be toxic by itself (Asano, 1980), and such internal bleeding leads to the following toxic events: elevated thrombin formation, increased extracellular glutamate level, red blood cell lysis, and iron toxicity (Hua et al., 2006; Wagner et al., 2006). Following damage to the microvasculature, there is a local reduction of blood flow resulting in different degrees of ischemia (Senter and Venes, 1978). In addition, the disruption of the BSCB and the resulting inflammatory response disturb the micro-environment and expose the adjacent tissue to molecules that can be noxious to non-injured tissue (Schlosshauer, 1993). The importance and a description of hemorrhage following SCI has been described previously (Mautes et al., 2000; Sinescu et al., 2010; Tator and Fehlings, 1991; Tator and Koyanagi, 1997). Notably, Tator and Koyanagi (1997) examined the distribution of hemorrhage following spinal cord injury in human. Their findings support the description that vascular damage is principally located in the intramedullary system, while the major arteria on the surface are usually spared. Thus hemorrhage after SCI is mostly distributed within the gray matter, which has proved difficult to model given that most injury models rely on the application of mechanical damage to the dorsal surface. Hitherto most accepted knowledge about the effect of hemorrhage in the spinal cord has been extrapolated from data from intracerebral hemorrhage (ICH) studies (Mautes et al., 2000), but such studies on the spinal cord have not been carried out. While the spinal cord is part of the CNS and shares many common characteristics with the brain, major immunological and anatomical differences do exist. An important example is the distinctive inflammatory response in the spinal cord compared to the brain following traumatic injury (Schnell et al., 1999). To assess the contribution of hemorrhage in traumatic spinal-cord injury, current mechanical models of spinal-cord injury using compression, contusion and surgical section are of limited value. A model is required which restricts the mechanical component of injury to the vessels. In the present study, microinjections of bacterial collagenase into the rat spinal cord were used in order to investigate the effect of intraspinal hemorrhage in the absence of severe mechanical injury. Bacterial collagenase digests type VI collagen in the basal lamina of blood vessels and has been used in established animal models of ICH (MacLellan et al., 2008). The following issues have been addressed: whether the degree of hemorrhage and blood-spinal-cord-barrier breakdown induced by collagenase injection is reproducible,
whether the degree of inflammatory response following hemorrhage in the spinal cord correlates with the level of hemorrhage, and whether there is evidence of neuronal and axonal damage. To observe the temporal sequence of events, three different time-points after the microinjection were chosen for the tissue collection: 6 h, 1 day and 7 days. We envisage that an improved understanding of the effect of hemorrhage on the spinal cord will aid clinical decisions in the acute setting, and may contribute to development of novel therapeutic interventions in the management of traumatic spinal cord injury.
2.
Results
2.1.
Collagenase disrupts the BSCB
Collagenase (0.12 U) or vehicle (saline) was microinjected into the spinal cord after laminectomy at T8. In order to examine the integrity of the blood-spinal-cord-barrier breakdown, we injected HRP intravenously 30 min before taking out the cords for immunohistochemistry. After processing, Hanker-Yates staining allowed us to quantify the extent, if any, of the damage done to the BSCB (Fig. 1a–c). In the animals injected with collagenase, we observed BSCB breakdown at 6 h (Po0.001) and 1 day after the microinjection (Po0.01). 7 Days after collagenase injection, a small amount of BSCB breakdown was still visible in some animals, but the difference compared to the vehicle-microinjected animals did not reach statistical significance (P ¼0.074). Little or no damage to the BSCB was observed in animals microinjected with vehicle at any time point. Any low-level damage found is probably attributable to the mechanical insertion of the microcapillary used for the injection.
2.2.
Collagenase leads to intraspinal hemorrhage
Following the loss of BSCB integrity due to the collagenase injection, we could observe an intraspinal hemorrhage, which was visible under the microscope as a brown-pigmented area (Fig. 1d–f). The pattern of the intraspinal hemorrhage measured was similar to that of the loss of BSCB integrity. Evident hemorrhage was observed 6 h (Po0.001) and 1 day (Po0.05) after the microinjection of collagenase, but none was detected after 7 days. In the vehicle-injected animals, little, if any, hemorrhage was observed at any time-point.
2.3.
Neutrophil profile in response to hemorrhage
There is no resident population of neutrophils in the spinal cord, and we were interested to discover whether hemorrhage caused by the microinjection of collagenase into the cord was sufficient to locally recruit neutrophils. Significant numbers of neutrophils were observed at 6 h after the injection of collagenase (Fig. 2a–c, Po0.001), and their magnitude remained high at 1 day (Po0.05). After 7 days, the number of neutrophils was still elevated in all the animals injected with collagenase compared to those injected with saline, but the numbers were low and the result did not reach significance. Some of the neutrophils observed in the spinal cord could be
Please cite this article as: Losey, P., et al., The role of hemorrhage following spinal-cord injury. Brain Research (2014), http: //dx.doi.org/10.1016/j.brainres.2014.04.033
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HRP area (mm2)
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Fig. 1 – Collagenase induced blood–spinal cord barrier breakdown and hemorrhage in the spinal cord. (a) Quantification of the HRP-positive (brown precipitate) areas after intraspinal microinjection of saline vehicle or collagenase. Representative transversal sections of the spinal cord 6 h after (b) vehicle or (c) collagenase microinjection. (d) Quantification of the blood load (margin of erythrocyte accumulation) after intraspinal microinjection of collagenase or saline control. Representative photomicrographs of transverse sections of the spinal cord 6 h after (e) vehicle or (f) collagenase microinjection (inset image is a high power of the margin of the bleed). Asterisk denotes statistical significance (n: Po0.05; nn: Po0.01 and nnn: Po0.001).
circulating neutrophils that enter the tissue due to the hemorrhage itself. However, it is to notice that the large number of neutrophils we observed could not solely be explained by the entry of circulating neutrophils and should be largely due to an active recruitment.
2.4.
ED1þve cells (recruited macrophages/activated microglia) was elevated in the spinal cord (Fig. 2d–f, Po0.01). Their Q3 number had increased markedly by 1 day (Po0.05), and further still by 7 days (Po0.05). Once again, we found little, if any, ED1þve cells in the saline-injected animals at any time-point.
Macrophage profile in response to hemorrhage 2.5.
The early transient increase in neutrophil recruitment in the collagenase-injected animals contrasted with a delayed, but sustained, increase in macrophage recruitment to the spinal cord. Six hours after collagenase injection, the number of
Neuronal integrity after the injection of collagenase
Quantification of the damaged area was done using Nissl staining. This method reveals RNA, and loss of Nissl staining Q4 is used as a lesion marker [35]. The presence of neuronal cell
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Fig. 2 – Neutrophils and macrophages. The number of positively stained neutrophils or macrophages were counted after the microinjection of saline vehicle control (open bars) or collagenase (solid bars) into the spinal cord at 6 h, 1 day, and 1 week after the microinjections. (a) Neutrophil numbers. Representative photomicrographs of sections immunostained for neutrophils 6 h after (b) vehicle or (c) collagenase. (d) Numbers of ED1-positive activated microglia/recruited monocytes. Photomicrographs of ED-1-positive cells 24 h after (e) vehicle or (f) collagenase. Asterisk denotes statistical significance (n: Po0.05; nn: Po0.01 and nnn: Po0.001). death was already revealed at 6 h (Fig. 3a–c; Po0.05) after the microinjection of collagenase. The extent of the injury was similar at the 1-day time-point (Po0.01). We found less evidence of damage at 7 days (Po0.05), which may be explained, at least partly, by shrinkage of the injured tissue. We found little or no damage at any time-point in the groups injected with saline.
2.6.
there was an increase of the number of injured axons. On-going axonal injury was less marked after 7 days than after 1 day, but it is important to note that axonal damage continues well beyond the period of breakdown of the BSCB, most probably due to axonal die-back (Coleman, 2005). A small number of APP-positive end-bulbs were visible in the saline-injected animals (Fig. 3e), but these were minor and were restricted to the microcapillary injection site.
Hemorrhage causes extended axonal damage 2.7.
Using APP immunohistochemistry, quantitative observation of collagenase-injected animals showed us that axonal damage was already present at 6 h (Fig. 3d–f). After 1 day
Locomotor functional deficits and loss of weight
Animals in the 7-day group were monitored daily for general health and wellbeing. The rats were active and no obvious
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Area of grey matter damage (mm2)
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50µm Fig. 3 – Neuronal and axonal damage. (a) Area of gray matter lesion. Photomicrographs of Nissl-stained sections from (b) vehicle or (c) collagenase injected cords. After collagenase injection, neuronal damage is evident in gray matter, visualized as an absence of cresyl violet stained neuronal cell. (d) Quantification of the number of damaged axons (APP-positive end-bulbs) after intraspinal microinjection of (e) vehicle or (f) collagenase. You can as well notice in the picture (d) that the extent of hemorrhage is visible even before processing the tissue. We can see in the picture of the cord significant effect of collagenase (Po0.001). Asterisk denotes statistical significance (n: Po0.05 and nn: Po0.01).
sickness behavior was noted in the follow-up period. The Basso, Beattie, Bresnahan (BBB) locomotor rating scale was used to document the locomotor function following microinjection. Normal spinal-cord function scores 21 while 0 represents total paralysis. All collagenase-injected animals had significant locomotor dysfunction (Fig. 4a; Po0.001), while no dysfunction was observed in the saline-injected group. The locomotor system was most affected during the first day after the collagenase injection, and progressively recovered over the following days. The weight of the collagenase-injected animals dropped slightly compared to the saline-injected animals at 1 day, but quickly returned to normal (Fig. 4b; Po0.001).
3.
Discussion
3.1.
Summary
In this study, we investigated the role of hemorrhage after spinal-cord injury. For the first time, we have been able to show that hemorrhage per se provokes an inflammatory response, and gives rise to axonal and neuronal damage and this structural damage gives rise to locomotor deficits. In this model hemorrhage was induced by the stereotaxic microinjection of collagenase into the gray matter of the spinal cord. By digesting type VI collagen in the basal lamina of blood vessels (MacLellan et al., 2008), collagenase produced a reproducible lesion that was associated with on-going
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Weight in %
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75 Vehicle Collagenase
50
25
0 d-1
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Fig. 4 – Locomotor deficits and weight loss. Early onset of locomotor dysfunction is observed in animals injected with 0.12 U collagenase when open field (a) locomotor function was evaluated using Basso, Beattie, Bresnahan locomotor scale. Significant interaction of treatment by time (Po0.0001). (b) Animals injected with collagenase show a slight reduction of weight as compared to saline vehicle animals. Significant interaction of treatment by time (Po0.0001).
axonal injury. It is important to note that the concentration of collagenase used in this study has been shown by others (Matsushita et al., 2000) not to be toxic in vitro when applied to neuronal cultures. We employed a low concentration of collagenase to minimize the likelihood of any direct neurotoxic effect versus true hemorrhage-induced pathophysiology.
3.2.
Hemorrhage after spinal-cord injury
Flanders demonstrated that the presence of hemorrhage in the spinal cord after spinal-cord injury is associated with significantly decreased motor function (Flanders et al., 1996). 85% of subjects with complete motor deficits have hemorrhagic lesions on their initial exams, and 21% of incomplete motor injuries have been found to have evidence of spinalcord hemorrhage. The extent of edema in the spinal cord after spinal-cord injury has also been associated with neurological outcome; the greater the extent of edema that is present in the spinal cord, the poorer the neurological outcome for a patient. The arteries that supply the spinal cord and those which are adjacent to the spinal column are at an increased risk of injury when spinal trauma occurs. The
arterial injuries associated with spinal trauma can result in devastating neurological deficits, but this rarely occurs. It is known that the vertebral arteries are frequently injured due to cervical spinal trauma, but that these injuries do not usually result in neurological deficits. However, intraparenchymal or intramedullary hemorrhage is usual after SCI and extends radially from the gray matter into the adjacent white matter. It is this type of bleeding that is thought to contribute most of the secondary damage associated with SCI. Indeed, in experimental models the early intraparenchymal distribution of hemorrhage coincides at later time points with the appearance of a cavity as a consequence of ischemia-induced necrosis, free radical damage, and inflammation (Mautes et al., 2000). Low blood flow adjacent to a hemorrhage site will also result in different levels of ischemia that might lead to delayed cell death. Intramedullary hemorrhage could also cause secondary damage by a direct compression of the adjacent structures by a mass effect or, downstream, by vasospasm due to the exposure to heme degradation products (Sinescu et al., 2010). Indeed, the iron content of heme can be an effective oxidant that even at low molecular concentrations is toxic to cultured cortical an spinal cord neurons (Regan and Panter, 1993; Regan and Guo, 1998). Hitherto it has been difficult to directly assess the effect of hemorrhage on spinal cord integrity in the absence of physical trauma. This is what the present model aims to provide.
3.3. A new model to study the impact of hemorrhage on spinal-cord injury The extent of hemorrhage and hemorrhagic necrosis has been shown to increase with the severity of an injury and is predictive of functional outcomes experimentally and clinically (Boldin et al., 2006). To date, there have been no models available to study the contribution of hemorrhage in isolation in the spinal cord. Here, we found that axonal damage is maximal 1 day after the induction of spinal hemorrhage and that axonal injury is not solely restricted to the initial bleed period; indeed, axonal injury is ongoing for at least 7 days after the bleed, and, as a consequence, the sequelae of hemorrhage after spinal injury are likely to be persistent and will continue to accumulate after the injury. It has been shown that the APP þve end bulbs persist for a relatively brief period. Following a stab injury, the number of end-bulbs rapidly declines over the first 48 h, indicating that, although some of the APPþ end-bulbs may persist for as long as 1 week, the majority are cleared, or resorbed by 72 h (Newman et al., 2001). It remains important to discover precisely how the presence of blood in the cord can give rise to this extended injury. It has been noted in contusion models that white matter injury continues to extend for up to one week postictus, and it has been suggested that the ongoing injury is likely due to clearance of tissue damaged by the primary impact rather than continuing cell death (Ek et al., 2010). Our results suggest that axonal damage can extend as a consequence of the presence of an intraparenchymal bleed in the absence of primary tissue damage as suggested by Ek et al. (2010). Following human spinal-cord injury hemorrhage is comparatively static (Leypold et al., 2008), but the on-going
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damage revealed by our studies suggests that active bleeding may not be the issue. It is clear that leukocyte recruitment is a feature of the bleed, but it remains unclear what mediators are induced that are responsible for their recruitment and whether the leukocytes are solely responsible for the damage or whether other blood-borne components, such as complement, may be responsible. The model described here will allow these issues to be investigated in an effective manner. Magnetic resonance imaging (MRI) has been used for the longitudinal assessment of progression of spinal-cord injury in rodents. Parameters that were highly correlated with histopathology were the quantity of hyperintense and hypointense signals, and lesion length. Importantly, it was the hypointense signal representing hemorrhage that was highly correlated to behavioral outcome measures (Mihai et al., 2008). We also found that the microinjection of collagenase generated behavioral deficits in our animals, although these were not sufficient to inhibit mobility or to significantly affect body weight over time. An issue that has hampered spinal-cord injury research has been the severity of the animal models that are used. In Western Europe, strict licensing requirements and emphasis on the three Rs (reduction, refinement, and replacement) has limited the use of spinal injury models. The present model is associated with mild clinical signs, which makes it attractive for studies on the role of hemorrhage on outcome and addresses the three Rs. Evidently this model does not replace other spinal cord models, since mechanical trauma is an important component of spinal cord injury, but it allows more detailed study of the role of hemorrhage following SCI. Interestingly, MRI has also revealed that the only effect of methylprednisolone (MP) administration was to decrease the extent of intramedullary spinal-cord hemorrhage in humans (Leypold et al., 2007). The effect was significant, but not large. Over the years there has been considerable debate over the usefulness of MP administration. MP does reduce neutrophil recruitment to the cord and a reduction in neutrophilinduced vascular dysfunction may be the mechanism by which MP reduces hemorrhage. Non-traumatic cerebral hemorrhage is an important cause of death and disability among adults. ICH accounts for approximately 10% of stroke cases, but the leading cause of death and disability after ICH is the ischemia that follows in the vascular beds that are served by the ruptured vessels. The collagenase-induced bleed will lead to an ischemic state (Lyden et al., 1997) in the cord which will be, in part, responsible for the damage we observed in this study. However, it should also be noted that the cerebral vessels involved are much larger than those affected in the cord, which will principally be at the arteriolar level. Collagenase injection into the basal ganglia of the rat induces learning and memory deficits that evolve over the course of several months. As was our experience the initial bleed gives rise to longer-term effects than expected (Hartman et al., 2008).
3.4. Inflammatory response following hemorrhage in the spinal cord In this study the microinjection of collagenase into the cord was associated with leukocyte recruitment. Others have
7
shown that the hematoma and parenchyma surrounding the hematoma are associated with white matter damage. Interestingly, axonal damage without demyelination was observed in that study after 3 days, at the edge of the hematoma and in the surrounding parenchyma, which is a region where we have shown there is no neuronal death (Wasserman and Schlichter, 2008). As the axonal damage preceded infiltration of activated microglia into the white matter tracts at 3 days, Wasserman concluded that the microglia cells respond to, rather than perpetrate, the damage. This laboratory has observed early axonal injury in white matter following endothelin-1-induced lesion that also occurs before significant microglial activation of recruitment (Hughes et al., 2003). Gao has addressed the role of microglia following ICH more directly (Gao et al., 2008). When adult male mice were subjected to ICH by intracaudate injection of either collagenase or autologous blood the elimination of monocytes/macrophages in CD11bHSVTK mice by ganciclovir resulted in reduced edema formation and an improvement in neurological outcomes. Interestingly, Wasserman found that minocycline, a metalloproteinase inhibitor and inhibitor of microglial activation, did reduce edema in the collagenase ICH model (Wasserman and Schlichter, 2007). The contribution of the neutrophils is likely to be particularly significant after spinal-cord hemorrhage where so many cells are recruited in this model. In the brain, many studies have indicated that leukocytes are a major contributor to brain injuries caused by ICH. Leukocyte-expressed CD18 is important for neutrophil-endothelial interactions in the vasculature. In CD18 ( / ) -knockout mice brain edema, as well as myeloperoxidase (MPO) activity and mortality, were significantly reduced following the collagenase-induced ICH (Titova et al., 2008). Locomotor recovery was observed in our new model and cannot all be attributed to the high plasticity of the rat nervous system, since this phenomenon needs more time to be effective (Darian-Smith, 2009; Hagg and Oudega, 2006). In addition to the reported axonal and neuronal damage, some of the dysfunction we observed will be a consequence of axonal conduction block caused the volume occupying effects of blood and edema, which will resolve more quickly. The contributions made by these elements after SCI can also be assessed more easily in this model compared to many others. Our laboratory and others (Neil-Dwyer and Cruickshank, 1974) have shown that subarachnoid hemorrhage is associated with raised blood total leukocyte and an acute-phase response (APR). Neutrophil counts are usually regarded as non-specific indicators of infection, inflammation, tissue necrosis, hemorrhage or stressful states. They can, however, be put to more specific uses, such as prognostic indicators in myocardial infarction (Hughes et al., 1963). It is tempting to speculate that the level of neutrophils in the circulation might serve as an additional indicator of hemorrhage in the cord and cases at risk. To investigate the role of the systemic APR, which is characterized by hepatic acute phase protein synthesis, leukocyte mobilization, fever, and changes in serum levels of glucocorticosteroids and cytokines (Gabay and Kushner, 1999), further experiments might be performed using this hemorrhage model.
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3.5.
Conclusion
In conclusion, we have now identified that damage to the vasculature that results in hemorrhage alone is likely play an important role in the outcome of spinal-cord injury. We expect that a better understanding of the mechanisms and molecules involved in these processes will have an impact on the development of novel treatment strategies for spinal-cord injury.
4.
Experimental procedure
4.1.
Animals
over 2 min and the capillary was left in place for a further 3 min before being slowly withdrawn. In vitro assays, with the same collagenase preparation at higher concentrations, have been show to be non-toxic to cultured neurons (Matsushita et al., 2000). The muscle layer was closed with 4/0 Vicryl (Johnson & Johnson, Ascot, UK) and the skin approximated with wound clips. After surgery, the animals recovered well and we did not observe evidence of overt distress or discomfort during the survival times of 6 h (n¼ 4 for both groups), 1 day (n¼4 for both groups) or 7 days (n¼ 3 for saline-injected rats and n ¼4 for collagenase-injected rats).
4.4.
2-Month-old male Wistar rats (180–210 g) (Harlan, UK) were used in the experiments. Animals were housed in groups of four or five in plastic cages with wood-chip bedding in specific-pathogen-free (SPF) facilities. Animals were kept under controlled temperature (2072 1C), maintained in a 12-h light/dark cycle, and supplied with food and water ad libitum. All procedures were carried out with Oxford University Research Ethics Committee approval under UK Home Office Licence 30/2008, in accordance with the Animals (Scientific Procedures) Act of 1986 (UK). All efforts were made to minimize animal suffering and distress.
4.2.
Reagents
Bacterial collagenase type IV from Colstridium histolyticum was obtained from Sigma-Aldrich (Poole, Dorset, UK). Collagenase was stored at 20 1C and diluted prior to injection with endotoxin-free saline. The dose of collagenase (0.12 U) used in this study was selected based on previous experiments, which used collagenase to model intracerebral hemorrhage in rats (MacLellan et al., 2004) and on pilot experiments.
4.3.
Surgical procedure
Animals were deeply anaesthetized with isoflurane (Rhodia Organique Fine Ltd., Bristol, UK) at 2.5–3% in oxygen for induction and maintenance. Aseptic surgery was carried out under an operating microscope (Wild M650, Leica, Milton Keynes, UK). Surgery was performed as previously described (Schnell et al., 1999). The skin overlying the thoracic spine was shaved and incised in the midline. Subcutaneous tissues and muscle layers were blunt-dissected and partial laminectomy was performed at thoracic level 8 (T8) to expose the underlying spinal cord without opening the dura mater. The animal was then suspended in the stereotactic frame by clamping of T7 and T9 spinous processes. The tip of a finely drawn calibrated glass capillary tube was stereotactically inserted into the gray matter of the spinal cord, 0.4 mm right-lateral of midline and at a depth of 1.6 mm. The details and advantages of this injection technique are described by McCluskey et al. (2008). The injection site was selected to ensure penetration into the spinal-cord parenchyma and for its proximity to the white matter of the lateral funiculus. 0.5 ml of collagenase (0.24 U/ml) or saline vehicle was injected
Perfusion and tissue collection
At appropriate survival times, rats were deeply anesthetized with an overdose of sodium pentobarbitone (Animal Care, Ltd., York, UK). Transcardiac perfusion was performed using heparinized saline, immediately followed by perfusion-fixing with 10% buffered formalin fixative. The spinal cord was removed and stored in 10% buffered formalin fixative for 7 days. A 2 cm-long section of the fixed spinal cord, incorporating the injection site at the center, was then cryo-protected in 30% sucrose for 2 days and frozen in Tissue-Tek (Sakura Finetek, Zoeterwoude, NL) for histology.
4.5.
Immunohistochemistry
20 mm-thick serial parasagittal sections of spinal cord were cut from frozen tissue blocks in the cryostat at 18 1C and mounted on APS-coated slides. All the histological evaluation was performed by investigators blind to the identity of the tissue. In accordance with standard immunohistochemistry methods (Campbell et al., 2007), polymorphonuclear neutrophils and activated macrophages were identified using antiMBS-2 serum (prepared in our laboratory) and anti-ED-1 serum (Serotec, Oxford, UK) respectively (Anthony et al., 1998). Anti-MBS-2 antibodies recognize epitopes present on the majority of neutrophil populations, while anti-ED-1 antibodies bind to an epitope on the lysosomal membrane of activated macrophages and microglia (Robinson et al., 1986). The primary antibodies were detected using the relevant biotinylated IgG, amplified using ABC and revealed with DAB (Vector Laboratories, Peterborough, UK). Sections were counterstained with cresyl violet. For each animal, a representative 20 mm-long parasagittal section was divided into ten 2 mm-long segments and, in each segment, a field with a high density of stained cells was quantified (400 magnification, Nikon Labophot-2), and the average number of positive cells in the parasagittal section was calculated and expressed as number of cells per mm2.
4.6.
Assessment of BSCB breakdown
Thirty minutes prior to the end of appropriate survival times, the Wistar rats were anaesthetized with isoflurane and injected with 104 U/kg of type-II horseradish peroxidase (HRP) intravenously (Sigma-Aldrich). HRP extravasation into the spinal cord was used to assess changes in BSCB permeability (Anthony et al., 1997). Hanker-Yates staining of 20 mmthick serial parasagittal sections was used to localize HRP.
Please cite this article as: Losey, P., et al., The role of hemorrhage following spinal-cord injury. Brain Research (2014), http: //dx.doi.org/10.1016/j.brainres.2014.04.033
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The parasagittal section of maximal HRP extravasation in each rat was traced under a light microscope (Leitz Laborlux S) using the camera lucida technique on paper (A4 Digital Paper Plus, Xerox). The traced area was then cut out, weighed (0.001–210 g WA 210, Adam Equipment, York, UK), and calculated by comparison against the weight of a reference section of paper.
4.7.
Assessment of hemorrhage
Nissl staining of 20 mm-thick serial parasagittal sections was used to localize areas of hemorrhage, as reflected by areas of brown pigmentation. Parasagittal sections of maximal hemorrhage in each rat were traced using camera lucida and the area calculated as for HRP extravasation.
4.8.
Assessment of neuronal damage
Nissl staining of 20 mm-thick serial parasagittal sections was used to assess neuronal damage. Parasagittal sections of maximal damage in each rat were traced using camera lucida and the area calculated as for HRP extravasation.
4.9.
Axonal damage
In order to demonstrate axonal damage caused by collagenase-induced bleeding and subsequent events, we performed immunohistochemistry for beta-amyloid precursor protein (β-APP) on 20 mm-thick parasagittal spinal cord sections. Immunohistochemistry was carried out as previously described (Bramlett et al., 1997) using anti-β-APP primary antibody (0.5 mg/ml, dilution 1:500, Zymed, San Francisco, USA). Accumulation of β-APP in clusters of axon terminal end-bulbs has been shown to be a sensitive marker of axonal damage after traumatic CNS injury (Gentleman et al., 1993). For each animal, a representative 20 mm-long parasagittal section was divided into five 4 mm-long segments and, in each segment, two fields with a high density of stained axonal bulbs was quantified (400 magnification, Nikon Labophot-2) and the density expressed as number of cells per mm2.
4.10.
Locomotor testing
Animals assigned to the 7-day survival group were assessed for open-field locomotor function using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale (Basso et al., 1995). Testing was performed at the same time each post-operative day by the same observer in a 100 100 cm2 black wooden box with 18 cm-high sidewalls. The observer had previous experience with the BBB locomotor rating scale and was blinded to the treatment received by the rats.
4.11.
Statistical analysis
Data were presented as mean7standard error of the mean (SEM). Comparison between different doses of collagenase and saline vehicle control for each survival time were assessed by analysis of variance (ANOVA) with Fisher's PLSD post hoc test. Differences between groups were accepted
9
as significant at the 95% confidence level (Po0.05). The BBB locomotor rating scale was analyzed using the Mann– Whitney U-test for non-parametric data.
4.12.
Statement of ethics
We certify that all applicable institutional and governmental regulations concerning the ethical use of animals were followed during the course of this research.
Conflict of interest No conflict of interest has been identified.
Acknowledgments P.L. is jointly supported by a Clarendon Award and a Greendale Scholarship (Merton College), University of Oxford, and a UK Universities Overseas Studentship Research Award and Biogen Idec Inc., Cambridge.
r e f e r e nc e s
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Research Report
The role of hemorrhage following spinal-cord injury$ Q1
Patrick Loseya,b,n, Christopher Youngc,1, Emily Krimholtza,1, Re´gis Bordetb,2, Daniel C. Anthonya,b,1 a
Experimental Neuropathology, Department of Pharmacology, University of Oxford, Oxford, UK EA 1046, Pharmacology, Faculty of Medicine, Research Branch, IMPRT, University of Lille North of France, Lille, France c Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK b
art i cle i nfo Article history: Accepted 23 April 2014
ab st rac t Q2 Spinal-cord injury is characterized by primary damage as a direct consequence of mechanical insult, and secondary damage that is partly due to the acute inflammatory response. The extent of any hemorrhage within the injured cord is also known to be
Keywords:
associated with the formation of intraparenchymal cavities and has been anecdotally
Spinal-cord injury
linked to secondary damage. This study was designed to examine the contribution of blood
Hemorrhage
components to the outcome of spinal-cord injury. We stereotaxically microinjected
Inflammation
collagenase, which causes localized bleeding, into the spinal cord to model the hemor-
Models of injury
rhage associated with spinal cord injury in the absence of significant mechanical trauma. Tissue damage was observed at the collagenase injection site over time, and was associated with localized disruption of the blood-spinal-cord barrier, neuronal cell death, and the recruitment of leukocytes. The magnitude of the bleed was related to neutrophil mobilization. Interestingly, the collagenase-induced injury also provoked extended axonal damage. With this model, the down-stream effects of hemorrhage are easily discernible, and the impact of treatment strategies for spinal-cord injury on hemorrhage-related injury can be evaluated. & 2014 Published by Elsevier B.V.
1.
Introduction
Traumatic spinal-cord injury (SCI) causes irreversible axonal damage and neuronal death, resulting in permanent disability. In addition to the initial mechanical injury, a cascade of events
takes place that precipitates further axonal damage and neuronal death long after the primary insult. This cascade of events is collectively termed secondary injury (Sekhon and Fehlings, 2001), and was first postulated as early as 1914 by Allen who suggested that noxious agents present in the
☆
Sources of support: This work was funded by the Medical Research Council under Grant number MRC G0300456. Corresponding author at: Experimental Neuropathology, Department of Pharmacology, University of Oxford, Oxford, UK. Fax: þ44 1865 271853. E-mail addresses: [email protected] (P. Losey), [email protected] (C. Young), [email protected] (E. Krimholtz), [email protected] (R. Bordet), [email protected] (D.C. Anthony). 1 Fax: þ44 1865 271853. 2 Fax: þ33 320446863. n
http://dx.doi.org/10.1016/j.brainres.2014.04.033 0006-8993/& 2014 Published by Elsevier B.V.
Please cite this article as: Losey, P., et al., The role of hemorrhage following spinal-cord injury. Brain Research (2014), http: //dx.doi.org/10.1016/j.brainres.2014.04.033
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hemorrhagic material might cause further damage to the spinal cord (Allen, 1914). The mechanisms of secondary injury include hemorrhage, tissue ischemia, blood-spinal-cordbarrier (BSCB) breakdown, inflammation, and glutamate toxicity, as well as demyelination and apoptotic signaling (Fleming et al., 2006). SCI is associated with mechanical insult to the microvasculature, leading to a primary hemorrhage into the spinal cord. Subsequent to the initial bleeding, there is a formation of secondary petechial hemorrhages in the tissue surrounding the primary lesion that have been shown to be related to a dramatic increase in the extent of the lesion (Gerzanich et al., 2009). Blood infiltrating the central nervous system (CNS) tissue has been shown to be toxic by itself (Asano, 1980), and such internal bleeding leads to the following toxic events: elevated thrombin formation, increased extracellular glutamate level, red blood cell lysis, and iron toxicity (Hua et al., 2006; Wagner et al., 2006). Following damage to the microvasculature, there is a local reduction of blood flow resulting in different degrees of ischemia (Senter and Venes, 1978). In addition, the disruption of the BSCB and the resulting inflammatory response disturb the micro-environment and expose the adjacent tissue to molecules that can be noxious to non-injured tissue (Schlosshauer, 1993). The importance and a description of hemorrhage following SCI has been described previously (Mautes et al., 2000; Sinescu et al., 2010; Tator and Fehlings, 1991; Tator and Koyanagi, 1997). Notably, Tator and Koyanagi (1997) examined the distribution of hemorrhage following spinal cord injury in human. Their findings support the description that vascular damage is principally located in the intramedullary system, while the major arteria on the surface are usually spared. Thus hemorrhage after SCI is mostly distributed within the gray matter, which has proved difficult to model given that most injury models rely on the application of mechanical damage to the dorsal surface. Hitherto most accepted knowledge about the effect of hemorrhage in the spinal cord has been extrapolated from data from intracerebral hemorrhage (ICH) studies (Mautes et al., 2000), but such studies on the spinal cord have not been carried out. While the spinal cord is part of the CNS and shares many common characteristics with the brain, major immunological and anatomical differences do exist. An important example is the distinctive inflammatory response in the spinal cord compared to the brain following traumatic injury (Schnell et al., 1999). To assess the contribution of hemorrhage in traumatic spinal-cord injury, current mechanical models of spinal-cord injury using compression, contusion and surgical section are of limited value. A model is required which restricts the mechanical component of injury to the vessels. In the present study, microinjections of bacterial collagenase into the rat spinal cord were used in order to investigate the effect of intraspinal hemorrhage in the absence of severe mechanical injury. Bacterial collagenase digests type VI collagen in the basal lamina of blood vessels and has been used in established animal models of ICH (MacLellan et al., 2008). The following issues have been addressed: whether the degree of hemorrhage and blood-spinal-cord-barrier breakdown induced by collagenase injection is reproducible,
whether the degree of inflammatory response following hemorrhage in the spinal cord correlates with the level of hemorrhage, and whether there is evidence of neuronal and axonal damage. To observe the temporal sequence of events, three different time-points after the microinjection were chosen for the tissue collection: 6 h, 1 day and 7 days. We envisage that an improved understanding of the effect of hemorrhage on the spinal cord will aid clinical decisions in the acute setting, and may contribute to development of novel therapeutic interventions in the management of traumatic spinal cord injury.
2.
Results
2.1.
Collagenase disrupts the BSCB
Collagenase (0.12 U) or vehicle (saline) was microinjected into the spinal cord after laminectomy at T8. In order to examine the integrity of the blood-spinal-cord-barrier breakdown, we injected HRP intravenously 30 min before taking out the cords for immunohistochemistry. After processing, Hanker-Yates staining allowed us to quantify the extent, if any, of the damage done to the BSCB (Fig. 1a–c). In the animals injected with collagenase, we observed BSCB breakdown at 6 h (Po0.001) and 1 day after the microinjection (Po0.01). 7 Days after collagenase injection, a small amount of BSCB breakdown was still visible in some animals, but the difference compared to the vehicle-microinjected animals did not reach statistical significance (P ¼0.074). Little or no damage to the BSCB was observed in animals microinjected with vehicle at any time point. Any low-level damage found is probably attributable to the mechanical insertion of the microcapillary used for the injection.
2.2.
Collagenase leads to intraspinal hemorrhage
Following the loss of BSCB integrity due to the collagenase injection, we could observe an intraspinal hemorrhage, which was visible under the microscope as a brown-pigmented area (Fig. 1d–f). The pattern of the intraspinal hemorrhage measured was similar to that of the loss of BSCB integrity. Evident hemorrhage was observed 6 h (Po0.001) and 1 day (Po0.05) after the microinjection of collagenase, but none was detected after 7 days. In the vehicle-injected animals, little, if any, hemorrhage was observed at any time-point.
2.3.
Neutrophil profile in response to hemorrhage
There is no resident population of neutrophils in the spinal cord, and we were interested to discover whether hemorrhage caused by the microinjection of collagenase into the cord was sufficient to locally recruit neutrophils. Significant numbers of neutrophils were observed at 6 h after the injection of collagenase (Fig. 2a–c, Po0.001), and their magnitude remained high at 1 day (Po0.05). After 7 days, the number of neutrophils was still elevated in all the animals injected with collagenase compared to those injected with saline, but the numbers were low and the result did not reach significance. Some of the neutrophils observed in the spinal cord could be
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30
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Fig. 1 – Collagenase induced blood–spinal cord barrier breakdown and hemorrhage in the spinal cord. (a) Quantification of the HRP-positive (brown precipitate) areas after intraspinal microinjection of saline vehicle or collagenase. Representative transversal sections of the spinal cord 6 h after (b) vehicle or (c) collagenase microinjection. (d) Quantification of the blood load (margin of erythrocyte accumulation) after intraspinal microinjection of collagenase or saline control. Representative photomicrographs of transverse sections of the spinal cord 6 h after (e) vehicle or (f) collagenase microinjection (inset image is a high power of the margin of the bleed). Asterisk denotes statistical significance (n: Po0.05; nn: Po0.01 and nnn: Po0.001).
circulating neutrophils that enter the tissue due to the hemorrhage itself. However, it is to notice that the large number of neutrophils we observed could not solely be explained by the entry of circulating neutrophils and should be largely due to an active recruitment.
2.4.
ED1þve cells (recruited macrophages/activated microglia) was elevated in the spinal cord (Fig. 2d–f, Po0.01). Their Q3 number had increased markedly by 1 day (Po0.05), and further still by 7 days (Po0.05). Once again, we found little, if any, ED1þve cells in the saline-injected animals at any time-point.
Macrophage profile in response to hemorrhage 2.5.
The early transient increase in neutrophil recruitment in the collagenase-injected animals contrasted with a delayed, but sustained, increase in macrophage recruitment to the spinal cord. Six hours after collagenase injection, the number of
Neuronal integrity after the injection of collagenase
Quantification of the damaged area was done using Nissl staining. This method reveals RNA, and loss of Nissl staining Q4 is used as a lesion marker [35]. The presence of neuronal cell
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Fig. 2 – Neutrophils and macrophages. The number of positively stained neutrophils or macrophages were counted after the microinjection of saline vehicle control (open bars) or collagenase (solid bars) into the spinal cord at 6 h, 1 day, and 1 week after the microinjections. (a) Neutrophil numbers. Representative photomicrographs of sections immunostained for neutrophils 6 h after (b) vehicle or (c) collagenase. (d) Numbers of ED1-positive activated microglia/recruited monocytes. Photomicrographs of ED-1-positive cells 24 h after (e) vehicle or (f) collagenase. Asterisk denotes statistical significance (n: Po0.05; nn: Po0.01 and nnn: Po0.001). death was already revealed at 6 h (Fig. 3a–c; Po0.05) after the microinjection of collagenase. The extent of the injury was similar at the 1-day time-point (Po0.01). We found less evidence of damage at 7 days (Po0.05), which may be explained, at least partly, by shrinkage of the injured tissue. We found little or no damage at any time-point in the groups injected with saline.
2.6.
there was an increase of the number of injured axons. On-going axonal injury was less marked after 7 days than after 1 day, but it is important to note that axonal damage continues well beyond the period of breakdown of the BSCB, most probably due to axonal die-back (Coleman, 2005). A small number of APP-positive end-bulbs were visible in the saline-injected animals (Fig. 3e), but these were minor and were restricted to the microcapillary injection site.
Hemorrhage causes extended axonal damage 2.7.
Using APP immunohistochemistry, quantitative observation of collagenase-injected animals showed us that axonal damage was already present at 6 h (Fig. 3d–f). After 1 day
Locomotor functional deficits and loss of weight
Animals in the 7-day group were monitored daily for general health and wellbeing. The rats were active and no obvious
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50µm Fig. 3 – Neuronal and axonal damage. (a) Area of gray matter lesion. Photomicrographs of Nissl-stained sections from (b) vehicle or (c) collagenase injected cords. After collagenase injection, neuronal damage is evident in gray matter, visualized as an absence of cresyl violet stained neuronal cell. (d) Quantification of the number of damaged axons (APP-positive end-bulbs) after intraspinal microinjection of (e) vehicle or (f) collagenase. You can as well notice in the picture (d) that the extent of hemorrhage is visible even before processing the tissue. We can see in the picture of the cord significant effect of collagenase (Po0.001). Asterisk denotes statistical significance (n: Po0.05 and nn: Po0.01).
sickness behavior was noted in the follow-up period. The Basso, Beattie, Bresnahan (BBB) locomotor rating scale was used to document the locomotor function following microinjection. Normal spinal-cord function scores 21 while 0 represents total paralysis. All collagenase-injected animals had significant locomotor dysfunction (Fig. 4a; Po0.001), while no dysfunction was observed in the saline-injected group. The locomotor system was most affected during the first day after the collagenase injection, and progressively recovered over the following days. The weight of the collagenase-injected animals dropped slightly compared to the saline-injected animals at 1 day, but quickly returned to normal (Fig. 4b; Po0.001).
3.
Discussion
3.1.
Summary
In this study, we investigated the role of hemorrhage after spinal-cord injury. For the first time, we have been able to show that hemorrhage per se provokes an inflammatory response, and gives rise to axonal and neuronal damage and this structural damage gives rise to locomotor deficits. In this model hemorrhage was induced by the stereotaxic microinjection of collagenase into the gray matter of the spinal cord. By digesting type VI collagen in the basal lamina of blood vessels (MacLellan et al., 2008), collagenase produced a reproducible lesion that was associated with on-going
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*
*
*
d0
d1
d2
d3
*
*
d2
d3
BBB score
21
14
7
0 d-1
d4
d5
d6
d4
d5
d6
125
100
Weight in %
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75 Vehicle Collagenase
50
25
0 d-1
d0
d1
Fig. 4 – Locomotor deficits and weight loss. Early onset of locomotor dysfunction is observed in animals injected with 0.12 U collagenase when open field (a) locomotor function was evaluated using Basso, Beattie, Bresnahan locomotor scale. Significant interaction of treatment by time (Po0.0001). (b) Animals injected with collagenase show a slight reduction of weight as compared to saline vehicle animals. Significant interaction of treatment by time (Po0.0001).
axonal injury. It is important to note that the concentration of collagenase used in this study has been shown by others (Matsushita et al., 2000) not to be toxic in vitro when applied to neuronal cultures. We employed a low concentration of collagenase to minimize the likelihood of any direct neurotoxic effect versus true hemorrhage-induced pathophysiology.
3.2.
Hemorrhage after spinal-cord injury
Flanders demonstrated that the presence of hemorrhage in the spinal cord after spinal-cord injury is associated with significantly decreased motor function (Flanders et al., 1996). 85% of subjects with complete motor deficits have hemorrhagic lesions on their initial exams, and 21% of incomplete motor injuries have been found to have evidence of spinalcord hemorrhage. The extent of edema in the spinal cord after spinal-cord injury has also been associated with neurological outcome; the greater the extent of edema that is present in the spinal cord, the poorer the neurological outcome for a patient. The arteries that supply the spinal cord and those which are adjacent to the spinal column are at an increased risk of injury when spinal trauma occurs. The
arterial injuries associated with spinal trauma can result in devastating neurological deficits, but this rarely occurs. It is known that the vertebral arteries are frequently injured due to cervical spinal trauma, but that these injuries do not usually result in neurological deficits. However, intraparenchymal or intramedullary hemorrhage is usual after SCI and extends radially from the gray matter into the adjacent white matter. It is this type of bleeding that is thought to contribute most of the secondary damage associated with SCI. Indeed, in experimental models the early intraparenchymal distribution of hemorrhage coincides at later time points with the appearance of a cavity as a consequence of ischemia-induced necrosis, free radical damage, and inflammation (Mautes et al., 2000). Low blood flow adjacent to a hemorrhage site will also result in different levels of ischemia that might lead to delayed cell death. Intramedullary hemorrhage could also cause secondary damage by a direct compression of the adjacent structures by a mass effect or, downstream, by vasospasm due to the exposure to heme degradation products (Sinescu et al., 2010). Indeed, the iron content of heme can be an effective oxidant that even at low molecular concentrations is toxic to cultured cortical an spinal cord neurons (Regan and Panter, 1993; Regan and Guo, 1998). Hitherto it has been difficult to directly assess the effect of hemorrhage on spinal cord integrity in the absence of physical trauma. This is what the present model aims to provide.
3.3. A new model to study the impact of hemorrhage on spinal-cord injury The extent of hemorrhage and hemorrhagic necrosis has been shown to increase with the severity of an injury and is predictive of functional outcomes experimentally and clinically (Boldin et al., 2006). To date, there have been no models available to study the contribution of hemorrhage in isolation in the spinal cord. Here, we found that axonal damage is maximal 1 day after the induction of spinal hemorrhage and that axonal injury is not solely restricted to the initial bleed period; indeed, axonal injury is ongoing for at least 7 days after the bleed, and, as a consequence, the sequelae of hemorrhage after spinal injury are likely to be persistent and will continue to accumulate after the injury. It has been shown that the APP þve end bulbs persist for a relatively brief period. Following a stab injury, the number of end-bulbs rapidly declines over the first 48 h, indicating that, although some of the APPþ end-bulbs may persist for as long as 1 week, the majority are cleared, or resorbed by 72 h (Newman et al., 2001). It remains important to discover precisely how the presence of blood in the cord can give rise to this extended injury. It has been noted in contusion models that white matter injury continues to extend for up to one week postictus, and it has been suggested that the ongoing injury is likely due to clearance of tissue damaged by the primary impact rather than continuing cell death (Ek et al., 2010). Our results suggest that axonal damage can extend as a consequence of the presence of an intraparenchymal bleed in the absence of primary tissue damage as suggested by Ek et al. (2010). Following human spinal-cord injury hemorrhage is comparatively static (Leypold et al., 2008), but the on-going
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damage revealed by our studies suggests that active bleeding may not be the issue. It is clear that leukocyte recruitment is a feature of the bleed, but it remains unclear what mediators are induced that are responsible for their recruitment and whether the leukocytes are solely responsible for the damage or whether other blood-borne components, such as complement, may be responsible. The model described here will allow these issues to be investigated in an effective manner. Magnetic resonance imaging (MRI) has been used for the longitudinal assessment of progression of spinal-cord injury in rodents. Parameters that were highly correlated with histopathology were the quantity of hyperintense and hypointense signals, and lesion length. Importantly, it was the hypointense signal representing hemorrhage that was highly correlated to behavioral outcome measures (Mihai et al., 2008). We also found that the microinjection of collagenase generated behavioral deficits in our animals, although these were not sufficient to inhibit mobility or to significantly affect body weight over time. An issue that has hampered spinal-cord injury research has been the severity of the animal models that are used. In Western Europe, strict licensing requirements and emphasis on the three Rs (reduction, refinement, and replacement) has limited the use of spinal injury models. The present model is associated with mild clinical signs, which makes it attractive for studies on the role of hemorrhage on outcome and addresses the three Rs. Evidently this model does not replace other spinal cord models, since mechanical trauma is an important component of spinal cord injury, but it allows more detailed study of the role of hemorrhage following SCI. Interestingly, MRI has also revealed that the only effect of methylprednisolone (MP) administration was to decrease the extent of intramedullary spinal-cord hemorrhage in humans (Leypold et al., 2007). The effect was significant, but not large. Over the years there has been considerable debate over the usefulness of MP administration. MP does reduce neutrophil recruitment to the cord and a reduction in neutrophilinduced vascular dysfunction may be the mechanism by which MP reduces hemorrhage. Non-traumatic cerebral hemorrhage is an important cause of death and disability among adults. ICH accounts for approximately 10% of stroke cases, but the leading cause of death and disability after ICH is the ischemia that follows in the vascular beds that are served by the ruptured vessels. The collagenase-induced bleed will lead to an ischemic state (Lyden et al., 1997) in the cord which will be, in part, responsible for the damage we observed in this study. However, it should also be noted that the cerebral vessels involved are much larger than those affected in the cord, which will principally be at the arteriolar level. Collagenase injection into the basal ganglia of the rat induces learning and memory deficits that evolve over the course of several months. As was our experience the initial bleed gives rise to longer-term effects than expected (Hartman et al., 2008).
3.4. Inflammatory response following hemorrhage in the spinal cord In this study the microinjection of collagenase into the cord was associated with leukocyte recruitment. Others have
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shown that the hematoma and parenchyma surrounding the hematoma are associated with white matter damage. Interestingly, axonal damage without demyelination was observed in that study after 3 days, at the edge of the hematoma and in the surrounding parenchyma, which is a region where we have shown there is no neuronal death (Wasserman and Schlichter, 2008). As the axonal damage preceded infiltration of activated microglia into the white matter tracts at 3 days, Wasserman concluded that the microglia cells respond to, rather than perpetrate, the damage. This laboratory has observed early axonal injury in white matter following endothelin-1-induced lesion that also occurs before significant microglial activation of recruitment (Hughes et al., 2003). Gao has addressed the role of microglia following ICH more directly (Gao et al., 2008). When adult male mice were subjected to ICH by intracaudate injection of either collagenase or autologous blood the elimination of monocytes/macrophages in CD11bHSVTK mice by ganciclovir resulted in reduced edema formation and an improvement in neurological outcomes. Interestingly, Wasserman found that minocycline, a metalloproteinase inhibitor and inhibitor of microglial activation, did reduce edema in the collagenase ICH model (Wasserman and Schlichter, 2007). The contribution of the neutrophils is likely to be particularly significant after spinal-cord hemorrhage where so many cells are recruited in this model. In the brain, many studies have indicated that leukocytes are a major contributor to brain injuries caused by ICH. Leukocyte-expressed CD18 is important for neutrophil-endothelial interactions in the vasculature. In CD18 ( / ) -knockout mice brain edema, as well as myeloperoxidase (MPO) activity and mortality, were significantly reduced following the collagenase-induced ICH (Titova et al., 2008). Locomotor recovery was observed in our new model and cannot all be attributed to the high plasticity of the rat nervous system, since this phenomenon needs more time to be effective (Darian-Smith, 2009; Hagg and Oudega, 2006). In addition to the reported axonal and neuronal damage, some of the dysfunction we observed will be a consequence of axonal conduction block caused the volume occupying effects of blood and edema, which will resolve more quickly. The contributions made by these elements after SCI can also be assessed more easily in this model compared to many others. Our laboratory and others (Neil-Dwyer and Cruickshank, 1974) have shown that subarachnoid hemorrhage is associated with raised blood total leukocyte and an acute-phase response (APR). Neutrophil counts are usually regarded as non-specific indicators of infection, inflammation, tissue necrosis, hemorrhage or stressful states. They can, however, be put to more specific uses, such as prognostic indicators in myocardial infarction (Hughes et al., 1963). It is tempting to speculate that the level of neutrophils in the circulation might serve as an additional indicator of hemorrhage in the cord and cases at risk. To investigate the role of the systemic APR, which is characterized by hepatic acute phase protein synthesis, leukocyte mobilization, fever, and changes in serum levels of glucocorticosteroids and cytokines (Gabay and Kushner, 1999), further experiments might be performed using this hemorrhage model.
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3.5.
Conclusion
In conclusion, we have now identified that damage to the vasculature that results in hemorrhage alone is likely play an important role in the outcome of spinal-cord injury. We expect that a better understanding of the mechanisms and molecules involved in these processes will have an impact on the development of novel treatment strategies for spinal-cord injury.
4.
Experimental procedure
4.1.
Animals
over 2 min and the capillary was left in place for a further 3 min before being slowly withdrawn. In vitro assays, with the same collagenase preparation at higher concentrations, have been show to be non-toxic to cultured neurons (Matsushita et al., 2000). The muscle layer was closed with 4/0 Vicryl (Johnson & Johnson, Ascot, UK) and the skin approximated with wound clips. After surgery, the animals recovered well and we did not observe evidence of overt distress or discomfort during the survival times of 6 h (n¼ 4 for both groups), 1 day (n¼4 for both groups) or 7 days (n¼ 3 for saline-injected rats and n ¼4 for collagenase-injected rats).
4.4.
2-Month-old male Wistar rats (180–210 g) (Harlan, UK) were used in the experiments. Animals were housed in groups of four or five in plastic cages with wood-chip bedding in specific-pathogen-free (SPF) facilities. Animals were kept under controlled temperature (2072 1C), maintained in a 12-h light/dark cycle, and supplied with food and water ad libitum. All procedures were carried out with Oxford University Research Ethics Committee approval under UK Home Office Licence 30/2008, in accordance with the Animals (Scientific Procedures) Act of 1986 (UK). All efforts were made to minimize animal suffering and distress.
4.2.
Reagents
Bacterial collagenase type IV from Colstridium histolyticum was obtained from Sigma-Aldrich (Poole, Dorset, UK). Collagenase was stored at 20 1C and diluted prior to injection with endotoxin-free saline. The dose of collagenase (0.12 U) used in this study was selected based on previous experiments, which used collagenase to model intracerebral hemorrhage in rats (MacLellan et al., 2004) and on pilot experiments.
4.3.
Surgical procedure
Animals were deeply anaesthetized with isoflurane (Rhodia Organique Fine Ltd., Bristol, UK) at 2.5–3% in oxygen for induction and maintenance. Aseptic surgery was carried out under an operating microscope (Wild M650, Leica, Milton Keynes, UK). Surgery was performed as previously described (Schnell et al., 1999). The skin overlying the thoracic spine was shaved and incised in the midline. Subcutaneous tissues and muscle layers were blunt-dissected and partial laminectomy was performed at thoracic level 8 (T8) to expose the underlying spinal cord without opening the dura mater. The animal was then suspended in the stereotactic frame by clamping of T7 and T9 spinous processes. The tip of a finely drawn calibrated glass capillary tube was stereotactically inserted into the gray matter of the spinal cord, 0.4 mm right-lateral of midline and at a depth of 1.6 mm. The details and advantages of this injection technique are described by McCluskey et al. (2008). The injection site was selected to ensure penetration into the spinal-cord parenchyma and for its proximity to the white matter of the lateral funiculus. 0.5 ml of collagenase (0.24 U/ml) or saline vehicle was injected
Perfusion and tissue collection
At appropriate survival times, rats were deeply anesthetized with an overdose of sodium pentobarbitone (Animal Care, Ltd., York, UK). Transcardiac perfusion was performed using heparinized saline, immediately followed by perfusion-fixing with 10% buffered formalin fixative. The spinal cord was removed and stored in 10% buffered formalin fixative for 7 days. A 2 cm-long section of the fixed spinal cord, incorporating the injection site at the center, was then cryo-protected in 30% sucrose for 2 days and frozen in Tissue-Tek (Sakura Finetek, Zoeterwoude, NL) for histology.
4.5.
Immunohistochemistry
20 mm-thick serial parasagittal sections of spinal cord were cut from frozen tissue blocks in the cryostat at 18 1C and mounted on APS-coated slides. All the histological evaluation was performed by investigators blind to the identity of the tissue. In accordance with standard immunohistochemistry methods (Campbell et al., 2007), polymorphonuclear neutrophils and activated macrophages were identified using antiMBS-2 serum (prepared in our laboratory) and anti-ED-1 serum (Serotec, Oxford, UK) respectively (Anthony et al., 1998). Anti-MBS-2 antibodies recognize epitopes present on the majority of neutrophil populations, while anti-ED-1 antibodies bind to an epitope on the lysosomal membrane of activated macrophages and microglia (Robinson et al., 1986). The primary antibodies were detected using the relevant biotinylated IgG, amplified using ABC and revealed with DAB (Vector Laboratories, Peterborough, UK). Sections were counterstained with cresyl violet. For each animal, a representative 20 mm-long parasagittal section was divided into ten 2 mm-long segments and, in each segment, a field with a high density of stained cells was quantified (400 magnification, Nikon Labophot-2), and the average number of positive cells in the parasagittal section was calculated and expressed as number of cells per mm2.
4.6.
Assessment of BSCB breakdown
Thirty minutes prior to the end of appropriate survival times, the Wistar rats were anaesthetized with isoflurane and injected with 104 U/kg of type-II horseradish peroxidase (HRP) intravenously (Sigma-Aldrich). HRP extravasation into the spinal cord was used to assess changes in BSCB permeability (Anthony et al., 1997). Hanker-Yates staining of 20 mmthick serial parasagittal sections was used to localize HRP.
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The parasagittal section of maximal HRP extravasation in each rat was traced under a light microscope (Leitz Laborlux S) using the camera lucida technique on paper (A4 Digital Paper Plus, Xerox). The traced area was then cut out, weighed (0.001–210 g WA 210, Adam Equipment, York, UK), and calculated by comparison against the weight of a reference section of paper.
4.7.
Assessment of hemorrhage
Nissl staining of 20 mm-thick serial parasagittal sections was used to localize areas of hemorrhage, as reflected by areas of brown pigmentation. Parasagittal sections of maximal hemorrhage in each rat were traced using camera lucida and the area calculated as for HRP extravasation.
4.8.
Assessment of neuronal damage
Nissl staining of 20 mm-thick serial parasagittal sections was used to assess neuronal damage. Parasagittal sections of maximal damage in each rat were traced using camera lucida and the area calculated as for HRP extravasation.
4.9.
Axonal damage
In order to demonstrate axonal damage caused by collagenase-induced bleeding and subsequent events, we performed immunohistochemistry for beta-amyloid precursor protein (β-APP) on 20 mm-thick parasagittal spinal cord sections. Immunohistochemistry was carried out as previously described (Bramlett et al., 1997) using anti-β-APP primary antibody (0.5 mg/ml, dilution 1:500, Zymed, San Francisco, USA). Accumulation of β-APP in clusters of axon terminal end-bulbs has been shown to be a sensitive marker of axonal damage after traumatic CNS injury (Gentleman et al., 1993). For each animal, a representative 20 mm-long parasagittal section was divided into five 4 mm-long segments and, in each segment, two fields with a high density of stained axonal bulbs was quantified (400 magnification, Nikon Labophot-2) and the density expressed as number of cells per mm2.
4.10.
Locomotor testing
Animals assigned to the 7-day survival group were assessed for open-field locomotor function using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale (Basso et al., 1995). Testing was performed at the same time each post-operative day by the same observer in a 100 100 cm2 black wooden box with 18 cm-high sidewalls. The observer had previous experience with the BBB locomotor rating scale and was blinded to the treatment received by the rats.
4.11.
Statistical analysis
Data were presented as mean7standard error of the mean (SEM). Comparison between different doses of collagenase and saline vehicle control for each survival time were assessed by analysis of variance (ANOVA) with Fisher's PLSD post hoc test. Differences between groups were accepted
9
as significant at the 95% confidence level (Po0.05). The BBB locomotor rating scale was analyzed using the Mann– Whitney U-test for non-parametric data.
4.12.
Statement of ethics
We certify that all applicable institutional and governmental regulations concerning the ethical use of animals were followed during the course of this research.
Conflict of interest No conflict of interest has been identified.
Acknowledgments P.L. is jointly supported by a Clarendon Award and a Greendale Scholarship (Merton College), University of Oxford, and a UK Universities Overseas Studentship Research Award and Biogen Idec Inc., Cambridge.
r e f e r e nc e s
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