The use of alkaline phosphatase as a bone turnover marker after spinal cord injury: A scoping review of human and animal studies.

BACKGROUND: Serum alkaline phosphatase (ALP) is measured as an indicator of bone or liver disease. Bone-specific alkaline phosphatase (B-ALP) is an isoform of ALP found in the bone tissue which can predict fractures and heterotopic ossification. OBJECTIVE: The aim of this scoping review was to explore the current use of ALP and B-ALP in studies using humans or animal models of SCI, and to identify ways to advance future research using ALP and B-ALP as a bone marker after SCI. RESULTS: HUMAN STUDIES: 42 studies were included. The evidence regarding changes or differences in ALP levels in individuals with SCI compared to controls is conflicting. For example, a negative correlation between B-ALP and total femur BMD was observed in only one of three studies examining the association. B-ALP seemed to increase after administration of teriparatide, and to decrease after treatment with denosumab. The effects of exercise on ALP and B-ALP levels are heterogeneous and depend on the type of exercise performed. ANIMAL STUDIES: 11 studies were included. There is uncertainty regarding the response of ALP or B-ALP levels after SCI; levels increased after some interventions, including vibration protocols, curcumin supplementation, cycles in electromagnetic field or hyperbaric chamber. Calcitonin or bisphosphonate administration did not affect ALP levels. CONCLUSION: Researchers are encouraged to measure the bone-specific isoform of ALP rather than total ALP in future studies in humans of animal models of SCI.

Protocol for rapid onset of mobilisation in patients with traumatic spinal cord injury (PROMPT-SCI) study: a single-arm proof-of-concept trial of early in-bed leg cycling following acute traumatic spinal cord injury.

INTRODUCTION: Activity-based therapy (ABT) is an important aspect of rehabilitation following traumatic spinal cord injury (SCI). Unfortunately, it has never been adapted to acute care despite compelling preclinical evidence showing that it is safe and effective for promoting neurological recovery when started within days after SCI. This article provides the protocol for a study that will determine the feasibility and explore potential benefits of early ABT in the form of in-bed leg cycling initiated within 48 hours after the end of spinal surgery for SCI. METHODS AND ANALYSIS: PROMPT-SCI (protocol for rapid onset of mobilisation in patients with traumatic SCI) is a single-site single-arm proof-of-concept trial. Forty-five patients aged 18 years or older with a severe traumatic SCI (American Spinal Injury Association Impairment Scale grade A, B or C) from C0 to L2 undergoing spinal surgery within 48 hours of the injury will be included. Participants will receive daily 30 min continuous sessions of in-bed leg cycling for 14 consecutive days, initiated within 48 hours of the end of spinal surgery. The feasibility outcomes are: (1) absence of serious adverse events associated with cycling, (2) completion of 1 full session within 48 hours of spinal surgery for 90% of participants and (3) completion of 11 sessions for 80% of participants. Patient outcomes 6 weeks and 6 months after the injury will be measured using neurofunctional assessments, quality of life questionnaires and inpatient length of stay. Feasibility and patient outcomes will be analysed with descriptive statistics. Patient outcomes will also be compared with a matched historical cohort that has not undergone in-bed cycling using McNemar and Student's t-tests for binary and continuous outcomes, respectively. ETHICS AND DISSEMINATION: PROMPT-SCI is approved by the Research Ethics Board of the CIUSSS NIM. Recruitment began in April 2021. Dissemination strategies include publications in scientific journals and presentations at conferences. TRIAL REGISTRATION NUMBER: NCT04699474.

Dual-Viral Transduction Utilizing Highly Efficient Retrograde Lentivirus Improves Labeling of Long Propriospinal Neurons.

The nervous system coordinates pathways and circuits to process sensory information and govern motor behaviors. Mapping these pathways is important to further understand the connectivity throughout the nervous system and is vital for developing treatments for neuronal diseases and disorders. We targeted long ascending propriospinal neurons (LAPNs) in the rat spinal cord utilizing Fluoro-Ruby (FR) [10kD rhodamine dextran amine (RDA)], and two dual-viral systems. Dual-viral tracing utilizing a retrograde adeno-associated virus (retroAAV), which confers robust labeling in the brain, resulted in a small number of LAPNs being labeled, but dual-viral tracing using a highly efficient retrograde (HiRet) lentivirus provided robust labeling similar to FR. Additionally, dual-viral tracing with HiRet lentivirus and tracing with FR may preferentially label different subpopulations of LAPNs. These data demonstrate that dual-viral tracing in the spinal cord employing a HiRet lentivirus provides robust and specific labeling of LAPNs and emphasizes the need to empirically optimize viral systems to target specific neuronal population(s).

N-acetylcysteine amide preserves mitochondrial bioenergetics and improves functional recovery following spinal trauma.

Mitochondrial dysfunction is becoming a pivotal target for neuroprotective strategies following contusion spinal cord injury (SCI) and the pharmacological compounds that maintain mitochondrial function confer neuroprotection and improve long-term hindlimb function after injury. In the current study we evaluated the efficacy of cell-permeating thiol, N-acetylcysteine amide (NACA), a precursor of endogenous antioxidant glutathione (GSH), on mitochondrial function acutely, and long-term tissue sparing and hindlimb locomotor recovery following upper lumbar contusion SCI. Some designated injured adult female Sprague-Dawley rats (n=120) received either vehicle or NACA (75, 150, 300 or 600mg/kg) at 15min and 6h post-injury. After 24h the total, synaptic, and non-synaptic mitochondrial populations were isolated from a single 1.5cm spinal cord segment (centered at injury site) and assessed for mitochondrial bioenergetics. Results showed compromised total mitochondrial bioenergetics following acute SCI that was significantly improved with NACA treatment in a dose-dependent manner, with maximum effects at 300mg/kg (n=4/group). For synaptic and non-synaptic mitochondria, only 300mg/kg NACA dosage showed efficacy. Similar dosage (300mg/kg) also maintained mitochondrial GSH near normal levels. Other designated injured rats (n=21) received continuous NACA (150 or 300mg/kg/day) treatment starting at 15min post-injury for one week to assess long-term functional recovery over 6weeks post-injury. Locomotor testing and novel gait analyses showed significantly improved hindlimb function with NACA that were associated with increased tissue sparing at the injury site. Overall, NACA treatment significantly maintained acute mitochondrial bioenergetics and normalized GSH levels following SCI, and prolonged delivery resulted in significant tissue sparing and improved recovery of hindlimb function.

Bone loss following spinal cord injury in a rat model.

The current study was undertaken to follow the time course of bone loss in the proximal tibia of rats over several weeks following thoracic contusion spinal cord injury (SCI) of varying severity. It was hypothesized that bone loss would be more pronounced in the more severely injured animals, and that hindlimb weight bearing would help prevent bone loss. Twenty-six female Sprague-Dawley rats (200-225 g, 6-7 weeks old) received standard thoracic (T9) injuries at energies of 6.25, 12.5, 25, or 50 g-cm. The rats were scored weekly for hindlimb function during locomotion. At 0, 2 or 3, and 8 weeks, high-resolution micro-CT images of each right tibia were obtained. Mechanical indentation testing was done to measure the compressive strength of the cancellous bone structure. The 6.25 g-cm group showed near normal locomotion, the 12.5 and 25 g-cm groups showed the ability to frequently or occasionally generate weight-supported plantar steps, respectively, and the 50 g-cm group showed only movement without weight-supported plantar stepping. The 6.25, 12.5 and 25 g-cm groups remained at the same level of bone volume fraction (cancBV/TV=0.24±0.07), while the 50 g-cm group experienced severe bone loss (67%), resulting in significantly lower (p<0.05) bone volume fraction (cancBV/TV=0.11±0.05) at 8 weeks. Proximal tibia cancellous bone strength was reduced by approximately 50% in these severely injured rats. Instead of a linear proportionality between injury severity and bone loss, there appears to be a distinct functional threshold, marked by occasional weight-supported stepping, above which bone loss does not occur.

Transplantation of ciliary neurotrophic factor-expressing adult oligodendrocyte precursor cells promotes remyelination and functional recovery after spinal cord injury.

Demyelination contributes to the dysfunction after traumatic spinal cord injury (SCI). We explored whether the combination of neurotrophic factors and transplantation of adult rat spinal cord oligodendrocyte precursor cells (OPCs) could enhance remyelination and functional recovery after SCI. Ciliary neurotrophic factor (CNTF) was the most effective neurotrophic factor to promote oligodendrocyte (OL) differentiation and survival of OPCs in vitro. OPCs were infected with retroviruses expressing enhanced green fluorescent protein (EGFP) or CNTF and transplanted into the contused adult thoracic spinal cord 9 d after injury. Seven weeks after transplantation, the grafted OPCs survived and integrated into the injured spinal cord. The survival of grafted CNTF-OPCs increased fourfold compared with EGFP-OPCs. The grafted OPCs differentiated into adenomatus polyposis coli (APC(+)) OLs, and CNTF significantly increased the percentage of APC(+) OLs from grafted OPCs. Immunofluorescent and immunoelectron microscopic analyses showed that the grafted OPCs formed central myelin sheaths around the axons in the injured spinal cord. The number of OL-remyelinated axons in ventrolateral funiculus (VLF) or lateral funiculus (LF) at the injured epicenter was significantly increased in animals that received CNTF-OPC grafts compared with all other groups. Importantly, 75% of rats receiving CNTF-OPC grafts recovered transcranial magnetic motor-evoked potential and magnetic interenlargement reflex responses, indicating that conduction through the demyelinated axons in VLF or LF, respectively, was partially restored. More importantly, recovery of hindlimb locomotor function was significantly enhanced in animals receiving grafts of CNTF-OPCs. Thus, combined treatment with OPC grafts expressing CNTF can enhance remyelination and facilitate functional recovery after traumatic SCI.

Rolipram attenuates acute oligodendrocyte death in the adult rat ventrolateral funiculus following contusive cervical spinal cord injury.

Rolipram, an inhibitor of phosphodiesterase 4 (PDE4) proteins that hydrolyze cAMP, increases axonal regeneration following spinal cord injury (SCI). Recent evidence indicate that rolipram also protects against a multitude of apoptotic signals, many of which are implicated in secondary cell death post-SCI. In the present study, we used immunohistochemistry and morphometry to determine potential spinal cord targets of rolipram and to test its protective potential in rats undergoing cervical spinal cord contusive injury. We found that 3 PDE4 subtypes (PDE4A, B, D) were expressed by spinal cord oligodendrocytes. OX-42 immunopositive microglia only expressed the PDE4B subtype. Oligodendrocyte somata were quantified within the cervical ventrolateral funiculus, a white matter region critical for locomotion, at varying time points after SCI in rats receiving rolipram or vehicle treatments. We show that rolipram significantly attenuated oligodendrocyte death at 24 h post-SCI continuing through 72 h, the longest time point examined. These results demonstrate for the first time that spinal cord glial cells express PDE4 subtypes and that the PDE4 inhibitor rolipram protects oligodendrocytes from secondary cell death following contusive SCI. They also indicate that further investigations into neuroprotection and axonal regeneration with rolipram are warranted for treating SCI.

Optimizing stem cell grafting into the CNS.

Grafting neural stem cell to facilitate repair after central nervous system (CNS) injury is being used in many laboratories. The technical challenges of this approach include the ability to maintain the viability of the cells before grafting, to be minimally invasive with the grafting method so as to not do further damage to the host CNS, and to maintain optimal viability of the cells during the grafting process. We outline an approach to CNS stem cell grafting that has evolved in our laboratories over the past decade (1-7). The best approach to graft a given stem cell population is empirical, but we provide parameters with which to quickly delineate that approach.