Femke Streijger

Biomarkers for Severity of Spinal Cord Injury in the Cerebrospinal Fluid of Rats

One of the major challenges in management of spinal cord injury (SCI) is that the assessment of injury severity is often imprecise. Identification of reliable, easily quantifiable biomarkers that delineate the severity of the initial injury and that have prognostic value for the degree of functional recovery would significantly aid the clinician in the choice of potential treatments. To find such biomarkers we performed quantitative liquid chromatography-mass spectrometry (LC-MS/MS) analyses of cerebrospinal fluid (CSF) collected from rats 24 h after either a moderate or severe SCI. We identified a panel of 42 putative biomarkers of SCI, 10 of which represent potential biomarkers of SCI severity. Three of the candidate biomarkers, Ywhaz, Itih4, and Gpx3 were also validated by Western blot in a biological replicate of the injury. The putative biomarkers identified in this study may potentially be a valuable tool in the assessment of the extent of spinal cord damage.

Intermittent Fasting Improves Functional Recovery after Rat Thoracic Contusion Spinal Cord Injury

Spinal cord injury (SCI) often results in a loss of motor and sensory function. Currently there are no validated effective clinical treatments. Previously we found in rats that dietary restriction, in the form of every-other-day fasting (EODF), started prior to (pre-EODF), or after (post-EODF) an incomplete cervical SCI was neuroprotective, increased plasticity, and promoted motor recovery. Here we examined if EODF initiated prior to, or after, a T10 thoracic contusion injury would similarly lead to enhanced functional recovery compared to ad libitum feeding. Additionally, we tested if a group fed every day (pair-fed), but with the same degree of restriction as the EODF animals (∼25% calorie restricted), would also promote functional recovery, to examine if EODFs effect is due to overall calorie restriction, or is specific to alternating sequences of 24-h fasts and ad libitum eating periods. Behaviorally, both pre- and post-EODF groups exhibited better functional recovery in the regularity indexed BBB ambulatory assessment, along with several parameters of their walking pattern measured with the CatWalk device, compared to both the ad-libitium-fed group as well as the pair-fed group. Several histological parameters (intensity and symmetry of serotonin immunostaining caudal to the injury and gray matter sparing) correlated with functional outcome; however, no group differences were observed. Thus besides the beneficial effects of EODF after a partial cervical SCI, we now report that alternating periods of fasting (but not pair-fed) also promotes improved hindlimb locomotion after thoracic spinal cord contusion, demonstrating its robust effect in two different injury models.

Lack of neuroprotective effects of simvastatin and minocycline in a model of cervical spinal cord injury

Minocycline, a commonly prescribed tetracycline antibiotic, has shown promise as a potential therapeutic agent in animal models of numerous neurologic disorders such as amyotrophic lateral sclerosis, multiple sclerosis, Parkinsons disease, Huntingtons disease, stroke, and spinal cord injury (SCI). Simvastatin is one of many hydroxymethylglutaryl-coenzyme-A reductase inhibitors prescribed to lower cholesterol. These drugs are also known to reduce inflammation and oxidative stress, improve endothelial function, and modulate the immune system in stroke, traumatic brain injury, and SCI. As both drugs have translational potential, we evaluated their neuroprotective properties here in a clinically relevant model of contusive cervical spinal cord injury. Sprague-Dawley rats underwent a unilateral cervical contusion SCI at C5 and were randomized to receive: 1. Minocycline 90 mg/kg x 3 days, 2. Simvastatin 20 mg/kg x 7 days, 3. Simvastatin 20 mg/kg x 7 days then 5mg/kg x 35 days, or 4. Saline (Control). Behavioral recovery was assessed over 6 weeks using the horizontal ladder test, cylinder rearing test, modified Montoya staircase test and grooming test. Forepaw sensitivity was also assessed using the electronic von Frey Aesthesiometer. The corticospinal and rubrospinal tracts were traced and the spinal cords were harvested 7 weeks after injury. The extent of gray matter and white matter sparing and corticospinal and rubrospinal tract sprouting were evaluated in cross sections of the spinal cord. In the end, neither minocycline nor simvastatin treatment was associated with improved performance on the behavioral tests, as compared to saline controls. Performance on the horizontal ladder test, cylinder rearing test, and von Frey sensory test were similar among all groups. Animals treated for 42 days with simvastatin scored significantly higher in the grooming score compared to other groups, but retrieved significantly fewer pellets on the modified Montoya staircase test than control and minocycline treated animals. Histologically, there were no significant differences in white and gray matter sparing and in the extent of corticospinal and rubrospinal sprouting between the four groups. In conclusion, both minocycline and simvastatin failed to improve functional and histological recovery in our model of contusive cervical spinal cord injury.

Ketogenic Diet Improves Forelimb Motor Function after Spinal Cord Injury in Rodents

High fat, low carbohydrate ketogenic diets (KD) are validated non-pharmacological treatments for some forms of drug-resistant epilepsy. Ketones reduce neuronal excitation and promote neuroprotection. Here, we investigated the efficacy of KD as a treatment for acute cervical spinal cord injury (SCI) in rats. Starting 4 hours following C5 hemi-contusion injury animals were fed either a standard carbohydrate based diet or a KD formulation with lipid to carbohydrate plus protein ratio of 3:1. The forelimb functional recovery was evaluated for 14 weeks, followed by quantitative histopathology. Post-injury 3:1 KD treatment resulted in increased usage and range of motion of the affected forepaw. Furthermore, KD improved pellet retrieval with recovery of wrist and digit movements. Importantly, after returning to a standard diet after 12 weeks of KD treatment, the improved forelimb function remained stable. Histologically, the spinal cords of KD treated animals displayed smaller lesion areas and more grey matter sparing. In addition, KD treatment increased the number of glucose transporter-1 positive blood vessels in the lesion penumbra and monocarboxylate transporter-1 (MCT1) expression. Pharmacological inhibition of MCTs with 4-CIN (α-cyano-4-hydroxycinnamate) prevented the KD-induced neuroprotection after SCI, In conclusion, post-injury KD effectively promotes functional recovery and is neuroprotective after cervical SCI. These beneficial effects require the function of monocarboxylate transporters responsible for ketone uptake and link the observed neuroprotection directly to the function of ketones, which are known to exert neuroprotection by multiple mechanisms. Our data suggest that current clinical nutritional guidelines, which include relatively high carbohydrate contents, should be revisited.

Large animal and primate models of spinal cord injury for the testing of novel therapies

Large animal and primate models of spinal cord injury (SCI) are being increasingly utilized for the testing of novel therapies. While these represent intermediary animal species between rodents and humans and offer the opportunity to pose unique research questions prior to clinical trials, the role that such large animal and primate models should play in the translational pipeline is unclear. In this initiative we engaged members of the SCI research community in a questionnaire and round-table focus group discussion around the use of such models. Forty-one SCI researchers from academia, industry, and granting agencies were asked to complete a questionnaire about their opinion regarding the use of large animal and primate models in the context of testing novel therapeutics. The questions centered around how large animal and primate models of SCI would be best utilized in the spectrum of preclinical testing, and how much testing in rodent models was warranted before employing these models. Further questions were posed at a focus group meeting attended by the respondents. The group generally felt that large animal and primate models of SCI serve a potentially useful role in the translational pipeline for novel therapies, and that the rational use of these models would depend on the type of therapy and specific research question being addressed. While testing within these models should not be mandatory, the detection of beneficial effects using these models lends additional support for translating a therapy to humans. These models provides an opportunity to evaluate and refine surgical procedures prior to use in humans, and safety and bio-distribution in a spinal cord more similar in size and anatomy to that of humans. Our results reveal that while many feel that these models are valuable in the testing of novel therapies, important questions remain unanswered about how they should be used and how data derived from them should be interpreted.

Mice lacking brain-type creatine kinase activity show defective thermoregulation

The cytosolic brain-type creatine kinase and mitochondrial ubiquitous creatine kinase (CK-B and UbCKmit) are expressed during the prepubescent and adult period of mammalian life. These creatine kinase (CK) isoforms are present in neural cell types throughout the central and peripheral nervous system and in smooth muscle containing tissues, where they have an important role in cellular energy homeostasis. Here, we report on the coupling of CK activity to body temperature rhythm and adaptive thermoregulation in mice. With both brain-type CK isoforms being absent, the body temperature reproducibly drops ~1.0 degrees C below normal during every morning (inactive) period in the daily cycle. Facultative non-shivering thermogenesis is also impaired, since CK--/-- mice develop severe hypothermia during 24 h cold exposure. A relationship with fat metabolism was suggested because comparison of CK--/-- mice with wildtype controls revealed decreased weight gain associated with less white and brown fat accumulation and smaller brown adipocytes. Also, circulating levels of glucose, triglycerides and leptin are reduced. Extensive physiological testing and uncoupling protein1 analysis showed, however, that the thermogenic problems are not due to abnormal responsiveness of brown adipocytes, since noradrenaline infusion produced a normal increase of body temperature. Moreover, we demonstrate that the cyclic drop in morning temperature is also not related to altered rhythmicity with reduced locomotion, diminished food intake or increased torpor sensitivity. Although several integral functions appear altered when CK is absent in the brain, combined findings point into the direction of inefficient neuronal transmission as the dominant factor in the thermoregulatory defect.

Cerebrospinal Fluid Biomarkers To Stratify Injury Severity and Predict Outcome in Human Traumatic Spinal Cord Injury

Neurologic impairment after spinal cord injury (SCI) is currently measured and classified by functional examination. Biological markers that objectively classify injury severity and predict outcome would greatly facilitate efforts to evaluate acute SCI therapies. The purpose of this study was to determine how well inflammatory and structural proteins within the cerebrospinal fluid (CSF) of acute traumatic SCI patients predicted American Spinal Injury Association Impairment Scale (AIS) grade conversion and motor score improvement over 6 months. Fifty acute SCI patients (29 AIS A, 9 AIS B, 12 AIS C; 32 cervical, 18 thoracic) were enrolled and CSF obtained through lumbar intrathecal catheters to analyze interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, tau, S100β, and glial fibrillary acidic protein (GFAP) at 24 h post-injury. The levels of IL-6, tau, S100β, and GFAP were significantly different between patients with baseline AIS grades of A, B, or C. The levels of all proteins (IL-6, IL-8, MCP-1, tau, S100β, and GFAP) were significantly different between those who improved an AIS grade over 6 months and those who did not improve. Linear discriminant analysis modeling was 83% accurate in predicting AIS conversion. For AIS A patients, the concentrations of proteins such as IL-6 and S100β correlated with conversion to AIS B or C. Motor score improvement also was strongly correlated with the 24-h post-injury CSF levels of all six biomarkers. The analysis of CSF can provide valuable biological information about injury severity and recovery potential after acute SCI. Such biological markers may be valuable tools for stratifying individuals in acute clinical trials where variability in spontaneous recovery requires large recruitment cohorts for sufficient power.

Characterization of a cervical spinal cord hemicontusion injury in mice using the infinite horizon impactor.

The majority of clinical spinal cord injuries (SCIs) are contusive and occur at the cervical level of the spinal cord. Most scientists and clinicians agree that the preclinical evaluation of novel candidate treatments should include testing in a cervical SCI contusion model. Because mice are increasingly used because of the availability of genetically engineered lines, we characterized a novel cervical hemicontusion injury in mice using the Infinite Horizon Spinal Cord Impactor (Precisions Systems & Instrumentation, Lexington, KY). In the current study, C57BL/6 mice received a hemicontusion injury of 75 kilodynes with or without dwell time in an attempt to elicit a sustained moderate-to-severe motor deficit. Hemicontusion injuries without dwell time resulted in sustained deficits of the affected forepaw, as revealed by a 3-fold decrease in usage during rearing, a ∼50% reduction in grooming scores, and retrieval of significantly fewer pellets on the Montoya staircase test. Only minor transient deficits were observed in grasping force. CatWalk analysis revealed reduced paw-print size and swing speed of the affected forelimb. Added dwell time of 15 or 30 sec significantly worsened behavioral outcome, and mice demonstrated minimal ability of grasping, paw usage, and overground locomotion. Besides worsening of behavioral deficits, added dwell time also reduced residual white and gray matter at the epicenter and rostral-caudal to the injury, including on the contralateral side of the spinal cord. Taken together, we developed and characterized a new hemicontusion SCI model in mice that produces sufficient and sustained impairments in gross and skilled forelimb function and produced primarily unilateral functional deficits.

A contusive model of unilateral cervical spinal cord injury using the infinite horizon impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.(1) Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor. Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5° (animals rotated at 22.5°), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoyas staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing. The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

Differential Histopathological and Behavioral Outcomes Eight Weeks after Rat Spinal Cord Injury by Contusion, Dislocation, and Distraction Mechanisms

Abstract The objective of this study was to compare the long-term histological and behavioral outcomes after spinal cord injury (SCI) induced by one of three distinct biomechanical mechanisms: dislocation, contusion, and distraction. Thirty male Sprague-Dawley rats were randomized to incur a traumatic cervical SCI by one of these three clinically relevant mechanisms. The injured cervical spines were surgically stabilized, and motor function was assessed for the following 8 weeks. The spinal cords were then harvested for histologic analysis. Quantification of white matter sparing using Luxol fast blue staining revealed that dislocation injury caused the greatest overall loss of white matter, both laterally and along the rostrocaudal axis of the injured cord. Distraction caused enlarged extracellular spaces and structural alteration in the white matter but spared the most myelinated axons overall. Contusion caused the most severe loss of myelinated axons in the dorsal white matter. Immunohistochemistry for the neuronal marker NeuN combined with Fluoro Nissl revealed that the dislocation mechanism resulted in the greatest neuronal cell losses in both the ventral and dorsal horns. After the distraction injury mechanism, animals displayed no recovery of grip strength over time, in contrast to the animals subjected to contusion or dislocation injuries. After the dislocation injury mechanism, animals displayed no improvement in the grooming test, in contrast to the animals subjected to contusion or distraction injuries. These data indicate that different SCI mechanisms result in distinct patterns of histopathology and behavioral recovery. Understanding this heterogeneity may be important for the future development of therapeutic interventions that target specific neuropathology after SCI.