Eugene Y. Koh

Disrupted autophagy after spinal cord injury is associated with ER stress and neuronal cell death

Autophagy is a catabolic mechanism facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner. Autophagy flux is necessary for normal neuronal homeostasis and its dysfunction contributes to neuronal cell death in several neurodegenerative diseases. Elevated autophagy has been reported after spinal cord injury (SCI); however, its mechanism, cell type specificity and relationship to cell death are unknown. Using a rat model of contusive SCI, we observed accumulation of LC3-II-positive autophagosomes starting at posttrauma day 1. This was accompanied by a pronounced accumulation of autophagy substrate protein p62, indicating that early elevation of autophagy markers reflected disrupted autophagosome degradation. Levels of lysosomal protease cathepsin D and numbers of cathepsin-D-positive lysosomes were also decreased at this time, suggesting that lysosomal damage may contribute to the observed defect in autophagy flux. Normalization of p62 levels started by day 7 after SCI, and was associated with increased cathepsin D levels. At day 1 after SCI, accumulation of autophagosomes was pronounced in ventral horn motor neurons and dorsal column oligodendrocytes and microglia. In motor neurons, disruption of autophagy strongly correlated with evidence of endoplasmic reticulum (ER) stress. As autophagy is thought to protect against ER stress, its disruption after SCI could contribute to ER-stress-induced neuronal apoptosis. Consistently, motor neurons showing disrupted autophagy co-expressed ER-stress-associated initiator caspase 12 and cleaved executioner caspase 3. Together, these findings indicate that SCI causes lysosomal dysfunction that contributes to autophagy disruption and associated ER-stress-induced neuronal apoptosis.

Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts.

Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs) could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis)-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases like osteoarthritis, in vivo.

Outcomes of lumbopelvic fixation in the treatment of complex sacral fractures using minimally invasive surgical techniques

BACKGROUND CONTEXTnComplex sacral fractures with vertical and anterior pelvic ring instability treated with traditional fixation methods are associated with high rates of failure and poor clinical outcomes. Supplemental lumbopelvic fixation (LPF) has been applied for additional stability to help with fracture union.nnnPURPOSEnThe study aimed to determine whether minimally invasive LPF provides reliable fracture stability and acceptable complication rates in cases of complex sacral fractures.nnnSTUDY DESIGN/SETTINGnThis is a retrospective cohort study at a single level I trauma center.nnnPATIENT SAMPLEnThe sample includes 24 patients who underwent minimally invasive LPF for complex sacral fracture with or without associated pelvic ring injury.nnnOUTCOME MEASURESnReoperation for all causes, loss of fixation, surgical time, transfusion requirements, length of hospital stay, postoperative day at mobilization, and mortality were evaluated.nnnMETHODSnPatient charts from 2008 to 2014 were reviewed. Of the 32 patients who underwent minimally invasive LPF for complex sacral fractures, 24 (12 male, 12 female) met all inclusion and exclusion criteria. Outcome measures were assessed with a retrospective chart review and radiographic review. The authors did not receive external funding for this study.nnnRESULTSnAcute reoperation was 12%, and elective reoperation was 29%. Two (8%) patients returned to the operating room for infection, one (4.2%) required revision for instrumentation malposition, and seven (29%) underwent elective removal of instrumentation. No patient experienced failure of instrumentation or loss of correction. Average surgical time was 3.6 hours, blood loss was 180u2009mL, transfusion requirement was 2.1 units of packed red blood cells, and postoperative mobilization was on postoperative day 5. No mortalities occurred as a result of the minimally invasive LPF procedure.nnnCONCLUSIONSnCompared with historic reports of open LPF, our results demonstrate reliable maintenance of reduction and acceptable complication rates with minimally invasive LPF for complexsacral fractures. The benefits of minimally invasive LPF may be offset with increased elective reoperations for removal of instrumentation.

Outcomes of Multilevel Vertebrectomy for Spondylodiscitis

BACKGROUND CONTEXTnThe incidence of pyogenic vertebral osteomyelitis (PVO) continues to increase in the United States, highlighting the need to recognize unique challenges presented by these cases and develop effective methods of surgical management. To date, no prior research has focused on the outcomes of PVO requiring two or more contiguous corpectomies.nnnPURPOSEnTo describe our experience in the operative management of PVO in 56 consecutive patients who underwent multilevel corpectomies (≥2 vertebral bodies) via a combined approach.nnnSTUDY DESIGN/SETTINGnSingle institution retrospective cohort review between January 2002 and December 2015. All patients had been treated at an academic tertiary referral center by one of two fellowship-trained orthopedic spine surgeons.nnnPATIENT SAMPLEnPatient records were cross-referenced with International Classification of Diseases osteomyelitis codes and paravertebral abscess code. Inclusion criteria for the study were patients within the cohort who had adequate medical records for review, a minimum patient age of 18 years, active vertebral osteomyelitis as an indication for surgical intervention, a minimum of 1-year radiographic follow-up, and surgical intervention that included at least two complete vertebral corpectomies. Subsequently, 56 patients met the inclusion criteria and were reviewed for this retrospective analysis.nnnOUTCOME MEASURESnOutcomes of interest were readmission and reoperation rates related to treatment of PVO, 30-day and 1-year mortality rates, radiographic outcomes, perioperative complications, infection control, and length of stay.nnnMETHODSnAfter obtaining approval from the Institutional Review Board, retrospective review was performed on records of all adults with PVO refractory to standard nonoperative treatment who underwent complete corpectomy of two or more contiguous vertebrae at a single institution between January 2002 and December 2015. This study was not funded, and no potential conflict of interest-associated biases were present.nnnRESULTSnFifty-six patients were identified (63% men; mean age 56.8 years; mean radiographic follow-up 2.8 years). Median length of stay was 13 days with nearly half readmitted (47%) after a median of 222.5 days after surgery. Twelve (22%) posterior revisions were required after a median 54 days for infection, painful or failed hardware, proximal junction kyphosis, adjacent level disease, or extension of the fusion. Thirty-day and 1-year mortality rates were 7.14% and 19.6%, respectively, with an infectious etiology as the most common cause of death.nnnCONCLUSIONSnMultilevel vertebral corpectomy for treatment of refractory vertebral osteomyelitis is associated with relatively high rates of complications and mortality compared with historical controls for 1 or 2 level procedures. We found clinical resolution and absence of complications requiring return to the operating room in 75% of patients when complete extirpation of the involved vertebrae is achieved. Our findings suggest multilevel anterior corpectomies with posterior stabilization may be a reasonable surgical option when approaching patients with complicated spondylodiscitis.

What are the trends and demographics in sports-related pediatric spinal cord injuries?

ABSTRACT Objectives: Pediatric spinal cord injury (PSCI) is a devastating injury that can cause significant long-term consequences. The purpose of this study is to calculate and report the prevalence of PSCI, identify risk factors for sports-related PSCI, and evaluate associated factors. Methods: The data sets of the Healthcare Cost and Utilization Project (HCUP) Kids’ Inpatient Database (KID) from 2000–2012 were analyzed using ICD-9-CM external cause of injury codes to identify the mechanism of injury contributing to PSCI hospitalization. We then extracted demographic data on each admission including age, gender, race, and year of admission. We further stratified the data by sports-related cases of injury. Multivariate logistic regression analyses were used to identify independent risk factors. Results: Of our study population, 0.8% had a documented diagnosis of spinal cord injury (SCI). The most common documented external cause of injury code was motor vehicle accidents, representing roughly half of all cases in patients 0–9 years-old (p = 0.001). PSCI due to sports as an external cause of injury was more prevalent in patients 10–17 years old, and was especially prevalent in the 10–13 year-old age category in which sports-related PSCI reached a high of 25.6%. Risk factors for traumatic PSCI after a sports-related external cause included being of older age, male, and white. Conclusions: The prevalence of SCI increased with age. Given the popularity of youth sports in the United States, parents and sports officials should be aware of the increased risk of sports-related PSCI among patients 10–17 years old. Level of evidence: Level III, retrospective cohort study

The Efficacy of Liposomal Bupivacaine in Lumbar Spine Surgery

ABSTRACT Background: Postoperative pain management in spine surgery holds unique challenges. The purpose of this study is to determine if the local anesthetic liposomal bupivacaine (LB) reduces the total opioid requirement in the first 3 days following posterior lumbar decompression and fusion (PLDF) surgery for degenerative spondylosis. Methods: Fifty patients underwent PLDF surgery in a prospective randomized control pilot trial between August 2015 and October 2016 and were equally allocated to either a treatment (LB) or a control (saline) group. Assessments included the 72-hour postoperative opioid requirement normalized to 1 morphine milligram equivalent (MME), visual analog scale (VAS), and hospital length of stay. Results: LB did not significantly alter the 72-hour postoperative opioid requirement compared to saline (11.6 vs. 13.4 MME, P = .40). In a subgroup analysis, there was also no significant difference in opioid consumption among narcotic-naive patients with either LB or saline. Among narcotic tolerant patients, however, opioid consumption was higher with saline than LB (20.6 MME vs. 13.3 MME, P = .048). Additionally, pre- and postoperative VAS scores and hospital length of stay were not significantly different with either LB or saline. Conclusions: In the setting of PLDF surgery, LB injections did not significantly reduce the consumption of opioids in the first 3 postoperative days, nor did the hospital length of stay or VAS pain scores, compared to saline. However, LB could be beneficial in reducing the consumption of opioids in narcotic-tolerant populations. Level of Evidence: 2.

Can Liposomal Bupivacaine Be Safely Utilized in Elective Spine Surgery

Study Design: Single-blinded prospective randomized control trial. Objectives: To compare the incidence of adverse events (AEs) and hospital length of stay between patients who received liposomal bupivacaine (LB) versus a single saline injection, following posterior lumbar decompression and fusion surgery for degenerative spondylosis. Methods: From 2015 to 2016, 59 patients undergoing posterior lumbar decompression and fusion surgery were prospectively enrolled and randomized to receive either 60 mL injection of 266 mg LB or 60 mL of 0.9% sterile saline, intraoperatively. Outcome measures included the incidence of postoperative AEs and hospital length of stay. Results: The most common AEs in the treatment group were nausea (39.3%), emesis (18.1%), and hypotension (18.1%). Nausea (23%), constipation (19.2%), and urinary retention (15.3%) were most common in the control group. Patients who received LB had an increased risk of developing nausea (relative risk [RR] = 1.7; 95% confidence interval [CI] = 0.75-3.8), emesis (RR = 2.3; 95% CI = 0.51-10.7), and headaches (RR = 2.36; 95% CI = 0.26-21.4). Patients receiving LB had a decreased risk of developing constipation (RR = 0.78; 95% CI = 0.25-2.43), urinary retention (RR = 0.78; 95% CI = 0.21-2.85), and pruritus (RR = 0.78; 95% = 0.21-2.8) postoperatively. Relative risk values mentioned above failed to reach statistical significance. No significant difference in the hospital length of stay between both groups was found (3.9 vs 3.9 days; P = .92). Conclusion: Single-dose injections of LB to the surgical site prior to wound closure did not significantly increase or decrease the incidence or risk of developing AEs postoperatively. Furthermore, no significant difference was found in the hospital length of stay between both groups.

Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis

Necroptosis, a regulated necrosis pathway mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), is induced following spinal cord injury (SCI) and thought to contribute to neuronal and glial cell death. However, mechanisms leading to activation of necroptosis after SCI remain unclear. We have previously shown that autophagy, a catabolic pathway facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner, is inhibited following SCI in rats. Our current data confirm that inhibition of autophagy also occurs after thoracic contusive SCI in the mouse model, as indicated by accumulation of both the autophagosome marker, LC3-II and autophagy cargo protein, p62/SQSTM1. This was most pronounced in the ventral horn neurons and was caused by rapid inhibition of lysosomal function after SCI. Interestingly, RIPK1, RIPK3, and the necroptosis effector protein MLKL also rapidly accumulated after SCI and localized to neurons with disrupted autophagy, suggesting that these events may be related. To determine if lysosomal dysfunction could contribute to induction of necroptosis, we treated PC12 cells and primary rat cortical neurons with lysosomal inhibitors. This led to rapid accumulation of RIPK1 and RIPK3, confirming that they are normally degraded by the lysosomal pathway. In PC12 cells lysosomal inhibition also sensitized cells to necroptosis induced by tumor necrosis factor α (TNFα) and caspase inhibitor. Imaging studies confirmed that RIPK1 partially localized to lysosomes in both untreated and lysosomal inhibitor treated cells. Similarly, we detected presence of RIPK1, RIPK3 and MLKL in both cytosol and at lysosomes after SCI in vivo. Furthermore, stimulation of autophagy and lysosomal function with rapamycin treatment led to decreased accumulation of RIPK1 and attenuated cell death after SCI. These data suggest that lysosomal dysfunction after SCI may contribute to both inhibition of autophagy and sensitize cells to necroptosis by promoting RIPK1 and RIPK3 accumulation.