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JOURNAL/nrgr/04.03/01300535-202412000-00032/figure1/v/2024-04-08T165401Z/r/image-tiff For patients with chronic spinal cord injury, the conventional treatment is rehabilitation and treatment of spinal cord injury complications such as urinary tract infection, pressure sores, osteoporosis, and deep vein thrombosis. Surgery is rarely performed on spinal cord injury in the chronic phase, and few treatments have been proven effective in chronic spinal cord injury patients. Development of effective therapies for chronic spinal cord injury patients is needed. We conducted a randomized controlled clinical trial in patients with chronic complete thoracic spinal cord injury to compare intensive rehabilitation (weight-bearing walking training) alone with surgical intervention plus intensive rehabilitation. This clinical trial was registered at ClinicalTrials.gov (NCT02663310). The goal of surgical intervention was spinal cord detethering, restoration of cerebrospinal fluid flow, and elimination of residual spinal cord compression. We found that surgical intervention plus weight-bearing walking training was associated with a higher incidence of American Spinal Injury Association Impairment Scale improvement, reduced spasticity, and more rapid bowel and bladder functional recovery than weight-bearing walking training alone. Overall, the surgical procedures and intensive rehabilitation were safe. American Spinal Injury Association Impairment Scale improvement was more common in T7-T11 injuries than in T2-T6 injuries. Surgery combined with rehabilitation appears to have a role in treatment of chronic spinal cord injury patients., (Copyright © 2024 Copyright: © 2024 Neural Regeneration Research.)

Accumulating evidence suggests that gut microbes modulate brain plasticity via the bidirectional gut-brain axis and play a role in stroke rehabilitation. However, the microbial species alterations associated with stroke and their correlation with functional outcome measures following acute stroke remain unknown. Here we measure post-stroke gut dysbiosis and how it correlates with gut permeability and cognitive functions in 12 stroke participants, 18 controls with risk factors for stroke, and 12 controls without risk factors. Stool samples were used to measure the microbiome with whole genome shotgun sequencing and leaky gut markers. We genotyped APOE status and measured diet composition and motor, cognitive, and emotional status using NIH Toolbox. We used linear regression methods to identify gut microbial associations with cognitive and emotional assessments. We did not find significance differences between the two control groups. In contrast, the bacteria populations of the Stroke group were statistically dissimilar from the control groups. Relative abundance analysis revealed notable decreases in butyrate-producing microbial taxa, secondary bile acid-producing taxa, and equol-producing taxa. The Stroke group had higher levels of the leaky gut marker alpha-1-antitrypsin in the stool than either of the groups and several taxa including Roseburia species (a butyrate producer) were negatively correlated with alpha-1-antitrypsin. Stroke participants scored lower on memory testing than those in the two control groups. Stroke participants with more Roseburia performed better on the picture vocabulary task; more Bacteroides uniformis (a butyrate producer) and less Escherichia coli (a pro-inflammatory species) reported higher levels of self-efficacy. Intakes of fiber, fruit and vegetable were lower, but sweetened beverages were higher, in the Stroke group compared with controls. Vegetable consumption was correlated with many bacterial changes among the participants, but only the species Clostridium bolteae, a pro-inflammatory species, was significantly associated with stroke. Our findings indicate that stroke is associated with a higher abundance of proinflammatory species and a lower abundance of butyrate producers and secondary bile acid producers. These altered microbial communities are associated with poorer functional performances. Future studies targeting the gut microbiome should be developed to elucidate whether its manipulation could optimize rehabilitation and boost recovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Hammond, Powell, Green, Chlipala, Frank, Yackzan, Yanckello, Chang, Xing, Heil, Springer, Pennypacker, Stromberg, Sawaki and Lin.)

Aneurysmal subarachnoid hemorrhage (aSAH) is a high mortality hemorrhagic stroke that affects nearly 30,000 patients annually in the United States. Approximately 30% of aSAH patients die during initial hospitalization and those who survive often carry poor prognosis with one in five having permanent physical and/or cognitive disabilities. The poor outcome of aSAH can be the result of the initial catastrophic event or due to the many acute or delayed neurological complications, such as cerebral ischemia, hydrocephalus, and re-bleeding. Unfortunately, no effective biomarker exists to predict or diagnose these complications at a clinically relevant time point when neurologic injury can be effectively treated and managed. Recently, a number of studies have demonstrated that microRNAs (miRNAs) in extracellular biofluids are highly associated with aSAH and complications. Here we provide an overview of the current research on relevant human studies examining the correlation between miRNAs and aSAH complications and discuss the potential application of using miRNAs as biomarkers in aSAH management.

Approximately one-third of aneurysmal subarachnoid hemorrhage (aSAH) patients develop delayed cerebral vasospasm (DCV) 3-10 days after aneurysm rupture resulting in additional, permanent neurologic disability. Currently, no validated biomarker is available to determine the risk of DCV in aSAH patients. MicroRNAs (miRNAs) have been implicated in virtually all human diseases, including aSAH, and are found in extracellular biofluids including plasma and cerebrospinal fluid (CSF). We used a custom designed TaqMan Low Density Array miRNA panel to examine the levels of 47 selected brain and vasculature injury related miRNAs in CSF and plasma specimens collected from 31 patients with or without DCV at 3 and 7 days after aSAH, as well as from eight healthy controls. The analysis of the first 18-patient cohort revealed a striking differential expression pattern of the selected miRNAs in CSF and plasma of aSAH patients with DCV from those without DCV. Importantly, this differential expression was observed at the early time point (3 days after aSAH), before DCV event occurs. Seven miRNAs were identified as reliable DCV risk predictors along with a prediction model constructed based on an array of additional 19 miRNAs on the panel. These chosen miRNAs were then used to predict the risk of DCV in a separate, testing cohort of 15 patients. The accuracy of DCV risk prediction in the testing cohort reached 87%. The study demonstrates that our novel designed miRNA panel is an effective predictor of DCV risk and has strong applications in clinical management of aSAH patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Springer, Xie, Fardo and Hatton.)

Background: The number of Canadian Residency Matching Service (CaRMS) applicants ranking surgical specialties as their first choice has declined over the past 20 years; concurrently, there has been a reduction in the number of hours spent teaching undergraduate medical education (UGME) anatomy, particularly with cadaveric dissection. The aim of this study was to determine the factors that most influence selection of a surgical specialty, with specific focus on the impact of UGME anatomy training., Methods: A 36-item cross-sectional survey was designed by experts in medical education and distributed to all current surgical residents in Canada in October 2018. Responses were recorded on a 5-point Likert scale or by means of list ranking. We analyzed univariable outcomes with a t test for continuous outcomes and the χ2 test for dichotomous outcomes., Results: Of 1493 surgical residents, 228 responded to the survey (response rate 15.3%). Respondents reported experiences on core rotations and elective rotations, and access to a mentor as the most important factors in deciding to pursue a surgical residency. Anatomy training with or without cadaveric dissection was moderately influential in respondents' first-choice CaRMS discipline (mean Likert scale score 2.97 [standard deviation (SD) 1.34] and 2.87 [SD 1.26], respectively). General surgery residents' CaRMS applications were more likely to have been influenced by UGME anatomy training than the applications by residents in other surgical specialties (p < 0.001). The impact of UGME anatomy training did not vary between postgraduate years or between male and female residents., Conclusion: Canadian surgical residents' decision to apply to a surgical specialty did not seem to be strongly influenced by their UGME anatomy training, with or without cadaveric dissection, but, rather, by factors such as clinical experience and surgical mentorship. Further evaluation of groups that were more positively affected by their UGME anatomy training is warranted., Competing Interests: None declared., (© 2021 Joule Inc. or its licensors.)

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional gene expression and contribute to all aspects of cellular function. We previously reported that the activities of several mitochondria-enriched miRNAs regulating inflammation (i.e., miR-142-3p, miR-142-5p, and miR-146a) are altered in the hippocampus at 3-12 hours following a severe traumatic brain injury. In the present study, we investigated the temporal expression profile of these inflammatory miRNAs in mitochondria and cytosol fractions at more chronic post-injury times following severe controlled cortical impact injury in rats. In addition, several inflammatory genes were analyzed in the cytosol fractions. The analysis showed that while elevated levels were observed in cytoplasm, the mitochondria-enriched miRNAs, miR-142-3p and miR-142-5p continued to be significantly reduced in mitochondria from injured hippocampi for at least 3 days and returned to near normal levels at 7 days post-injury. Although not statistically significant, miR-146a also remained at reduced levels for up to 3 days following controlled cortical impact injury, and recovered by 7 days. In contrast, miRNAs that are not enriched in mitochondria, including miR-124a, miR-150, miR-19b, miR-155, and miR-223 were either increased or demonstrated no change in their levels in mitochondrial fractions for 7 days. The one exception was that miR-223 levels were reduced in mitochondria at 1 day following injury. No major alterations were observed in sham operated animals. This temporal pattern was unique to mitochondria-enriched miRNAs and correlated with injury-induced changes in mitochondrial bioenergetics as well as expression levels of several inflammatory markers. These observations suggested a potential compartmental re-distribution of the mitochondria-enriched inflammatory miRNAs and may reflect an intracellular mechanism by which specific miRNAs regulate injury-induced inflammatory signaling. To test this, we utilized a novel peptide-based nanoparticle strategy for in vitro and in vivo delivery of a miR-146a mimic as a potential therapeutic strategy for targeting nuclear factor-kappaB inflammatory modulators in the injured brain. Nanoparticle delivery of miR-146a to BV-2 or SH-SY5Y cells significantly reduced expression of TNF receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1), two important modulators of the nuclear factor-kappaB (NF-κB) pro-inflammatory pathway. Moreover, injections of miR-146a containing nanoparticles into the brain immediately following controlled cortical impact injury significantly reduced hippocampal TNF receptor-associated factor 6 and interleukin-1 receptor-associated kinase 1 levels. Taken together, our studies demonstrate the subcellular alteration of inflammatory miRNAs after traumatic brain injury and establish proof of principle that nanoparticle delivery of miR-146a has therapeutic potential for modulating pro-inflammatory effectors in the injured brain. All of the studies performed were approved by the University of Kentucky Institutional Animal Care and Usage Committee (IACUC protocol # 2014-1300) on August 17, 2017., Competing Interests: None

Background: The incidence of colorectal cancer in North America is rising among patients younger than 50 years. Available data are conflicting regarding presentation and outcomes in this population. This review aimed to synthesize literature regarding young patients with colorectal cancer with respect to patient demographics, disease extent and survival, compared with patients older than 50 years., Methods: We searched Medline, Embase, the Cochrane Central Register of Controlled Trials and PubMed for articles published between 1990 and the time of search. Articles comparing North American patients with colorectal cancer younger and older than 50 years were eligible for inclusion. We used a random-effects model to pool odds ratios., Results: Eight retrospective studies were eligible for inclusion (n = 790 959). Mean age was 42.6 years (standard deviation [SD] 5.07) in the younger group, and 69.1 years (SD 9.25) in the older group. Young patients were more likely to present with regional (odds ratio [OR] 1.27, 95% confidence interval [CI] 1.16-1.40) and distant disease (OR 1.47, 95%CI 1.30-1.67). Considering patients at all stages of disease, differences in 5-year overall survival (OR 1.54, 95%CI 0.96-2.47) and cancer-specific survival (OR 1.01, 95%CI 0.91-1.13) were not statistically significant between groups. However, when controlling for disease extent, 5-year cancer-specific survival was significantly higher among young patients with local (OR 1.69, 95%CI 1.43-1.99), regional (OR 1.37, 95%CI 1.16-1.63) and distant disease (OR 1.79, 95%CI 1.45-2.21)., Conclusion: North American patients presenting with colorectal cancer before the age of 50 years are more likely to have advanced disease. Although overall and cancer-specific survival is not significantly different between these groups, younger patients have improved survival when controlling for cancer stage., Competing Interests: None declared., (© 2021 Joule Inc. or its licensors.)

Background: Enhanced Recovery After Surgery (ERAS) protocols use evidence-based perioperative practices that reduce morbidity and length of stay and improve patient satisfaction. ERAS is considered standard of care; however, utilization remains low and substantial practice variation exists. The aim of this study was to pragmatically characterize variation in colorectal surgery practice and identify predictors of ERAS utilization., Methods: A survey of general surgeons identified using the Ontario College of Physicians and Surgeons database was conducted. Information on basic demographic characteristics, utilization of ERAS and predictors of ERAS implementation was collected. Nine ERAS behaviours were analyzed. Multivariable analysis was used to determine effects of demographic, hospital and surgeon covariates on ERAS utilization., Results: Seven hundred and ninety-seven general surgeons were invited to participate in the survey, and 235 general surgeons representing 84 Ontario hospitals responded (30% response rate). Surgeons practising in academic settings and in large community hospitals represented 30% and 47% of the respondents, respectively. A total of 20% of the respondents used all 9 ERAS behaviours consistently. Rates of diet advancement on postoperative day 0, intravenous fluid restriction and having catheter and line procedures were significantly higher among respondents who adhered to ERAS protocols than among those who did not (74% v. 54%, p = 0.004; 92% v. 80%, p = 0.01; and 91% v. 41%, p < 0.001, respectively). Respondents from academic settings reported practising nearly 1 more ERAS behaviour than those from small community hospitals (odds ratio [OR] 0.86, 95% confidence interval [CI] 0.42 to 1.31, p < 0.001). Multivariable analysis demonstrated that colorectal fellowship training or exposure to ERAS during training did not significantly affect ERAS behaviour utilization (OR 0.32, 95% CI -0.31 to 0.94, p = 0.16; OR 0.28, 95% CI -0.26 to 0.82, p = 0.16, respectively)., Conclusion: Substantial practice variation in colorectal surgery still exists. Individual ERAS principles are commonly followed; however, ERAS behaviours are not widely formalized into hospital protocols.

While mitochondria maintain essential cellular functions, such as energy production, calcium homeostasis, and regulating programmed cellular death, they also play a major role in pathophysiology of many neurological disorders. Furthermore, several neurodegenerative diseases are closely linked with synaptic damage and synaptic mitochondrial dysfunction. Unfortunately, the ability to assess mitochondrial dysfunction and the efficacy of mitochondrial-targeted therapies in experimental models of neurodegenerative disease and CNS injury is limited by current mitochondrial isolation techniques. Density gradient ultracentrifugation (UC) is currently the only technique that can separate synaptic and non-synaptic mitochondrial sub-populations, though small brain regions cannot be assayed due to low mitochondrial yield. To address this limitation, we used fractionated mitochondrial magnetic separation (FMMS), employing magnetic anti-Tom22 antibodies, to develop a novel strategy for isolation of functional synaptic and non-synaptic mitochondria from mouse cortex and hippocampus without the usage of UC. We compared the yield and functionality of mitochondria derived using FMMS to those derived by UC. FMMS produced 3x more synaptic mitochondrial protein yield compared to UC from the same amount of tissue, a mouse hippocampus. FMMS also has increased sensitivity, compared to UC separation, to measure decreased mitochondrial respiration, demonstrated in a paradigm of mild closed head injury. Taken together, FMMS enables improved brain-derived mitochondrial yield for mitochondrial assessments and better detection of mitochondrial impairment in CNS injury and neurodegenerative disease.

The mitochondrion serves many functions in the central nervous system (CNS) and other organs beyond the well-recognized role of adenosine triphosphate (ATP) production. This includes calcium-dependent cell signaling, regulation of gene expression, synthesis and release of cytotoxic reactive oxygen species, and the release of cytochrome c and other apoptotic cell death factors. Traumatic injury to the CNS results in a rapid and, in some cases, sustained loss of mitochondrial function. One consequence of compromised mitochondrial function is induction of the mitochondrial permeability transition (mPT) state due to formation of the cyclosporine A sensitive permeability transition pore (mPTP). In this mini-review, we summarize evidence supporting the involvement of the mPTP as a mediator of mitochondrial and cellular demise following CNS traumatic injury and discuss the beneficial effects and limitations of the current ex-perimental strategies targeting the mPTP., Competing Interests: None declared

Background: Postoperative ileus is a poorly understood multifactorial outcome following colorectal surgery that presents significant clinical challenges and contributes to increased morbidity, length of stay, and healthcare cost. To date, there are few pharmacological interventions that shorten the duration of postoperative ileus., Objective: This study is the first to evaluate the efficacy of simethicone in treating postoperative ileus symptoms in patients undergoing colorectal surgery., Design: A multicenter, double-blinded, placebo controlled randomized controlled trial., Settings: This trial was conducted at two academic tertiary care centres in Ontario, Canada., Participants: 118 patients undergoing colorectal surgery., Interventions: Patients were randomized to receive either a five-day course of oral simethicone (n = 58) or a placebo (n = 60)., Main Outcome Measures: The primary outcome was time to first passage of flatus. Secondary outcomes included time to first bowel movement, postoperative length of stay, and postoperative pain. Statistical analyses were performed on an intention-to-treat basis. Statistical significance set at p = 0.05., Results: The median time to first passage of flatus in simethicone arm was 25.2 h and 26.7 h in controls (P = 0.98). There were no significant differences in the median time to first bowel movement (simethicone = 41.1 h vs. control = 42.9 h, P = 0.91) or median length of hospital stay (simethicone = 4.5 days vs. control = 4.0 days, P = 0.63)., Conclusions: This study failed to show a difference in return of gastrointestinal motility in patients receiving simethicone following colorectal surgery. Postoperative ileus remains a significant clinical and economic burden to the healthcare system and further research is needed to identify a reliable and effective method of treatment., (Copyright © 2018 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.)

Mitochondrial homeostasis is essential for maintaining cellular function and survival in the central nervous system (CNS). Mitochondrial function is significantly compromised after spinal cord injury (SCI) and is associated with accumulation of high levels of calcium, increased production of free radicals, oxidative damage, and eventually mitochondrial permeability transition (mPT). The formation of the mPT pore (mPTP) and subsequent mPT state are considered to be end stage events in the decline of mitochondrial integrity, and strategies that inhibit mPT can limit mitochondrial demise. Cyclosporine A (CsA) is thought to inhibit mPT by binding to cyclophilin D and has been shown to be effective in models of CNS injury. CsA, however, also inhibits calcineurin, which is responsible for its immunosuppressive properties. In the present study, we conducted a dose-response examination of NIM811, a nonimmunosuppressive CsA analog, on recovery of function and tissue sparing in a rat model of moderate to severe SCI. The results of our experiments revealed that NIM811 (10 mg/kg) significantly improved open field locomotor performance, while the two higher doses tested (20 and 40 mg/kg) significantly improved return of reflexive bladder control and significantly decreased the rostral-caudal extent of the lesion. Taken together, these results demonstrate the ability of NIM811 to improve recovery of function in SCI and support the role of protecting mitochondrial function as a potential therapeutic target.

Cerebellar function is critical for coordinating movement and motor learning. However, events occurring in the cerebellum following spinal cord injury (SCI) have not been investigated in detail. We provide evidence of SCI-induced cerebellar synaptic changes involving a loss of granule cell parallel fiber input to distal regions of the Purkinje cell dendritic tree. This is accompanied by an apparent increase in synaptic contacts to Purkinje cell proximal dendrites, presumably from climbing fibers originating in the inferior olive. We also observed an early stage injury-induced decrease in the levels of cerebellin-1, a synaptic organizing molecule that is critical for establishing and maintaining parallel fiber-Purkinje cell synaptic integrity. Interestingly, this transsynaptic reorganizational pattern is consistent with that reported during development and in certain transgenic mouse models. To our knowledge, such a reorganizational event has not been described in response to SCI in adult rats. Regardless, the novel results of this study are important for understanding SCI-induced synaptic changes in the cerebellum, which may prove critical for strategies focusing on promoting functional recovery.

Objectives: To investigate the incidence of spinal abscess and substance abuse in a tertiary care hospital after state legislation titled "House Bill 1" (HB1) mandated stricter regulation of prescription drugs of abuse in Kentucky in 2012., Design: A retrospective case series study design was used to review the incidence of spinal abscess and drug abuse diagnoses admissions from 2010 to 2014. Variances in the incidence of spinal abscess and substance abuse were plotted across this time frame., Results: The incidence of intraspinal abscess increased 1.56-fold in 2011 (n = 26) and 2012 (n = 25) relative to 2010 (n = 16). However, in 2013, the year following implementation of HB1 legislation, the incidence of intraspinal abscess increased 2.38-fold (n = 38) and then 4.19-fold (n = 67) in 2014. The incidence of intraspinal abscess in subjects with drug abuse diagnosis remained constant between 2010 (n = 3) and 2012 (n = 3). However, it increased twofold (n = 7) in 2013 and then ninefold (n = 27) in 2014. A correlation coefficient (rSAD ) of 0.775 revealed a strong association between the increase incidence of intraspinal abscess and diagnosis of drug abuse., Conclusions: The results of this retrospective study demonstrate an increased incidence of intraspinal abscess associated with drug abuse after passage of HB1 legislation regulating prescriptions of controlled medications in Kentucky. This increased incidence may be related to individuals relying on nonprescription drugs of abuse due to more highly regulated access to controlled prescription medications. However, additional factors unrelated to HB1 legislation must be taken into account., (Wiley Periodicals, Inc.)

Background: The purpose of this research was to examine the morbidity, mortality and rate of recurrent bowel obstruction associated with the treatment of small bowel obstruction (SBO) in older adults., Methods: We prospectively enrolled all patients 70 years or older with an SBO who were admitted to a tertiary care teaching centre between Jul. 1, 2011, and Sept. 30, 2012. Data regarding presentation, investigations, treatment and outcomes were collected., Results: Of the 104 patients admitted with an SBO, 49% were managed nonoperatively and 51% underwent surgery. Patients who underwent surgery experienced more complications (64% v. 27%, p = 0.002) and stayed in hospital longer (10 v. 3 d, p < 0.001) than patients managed nonoperatively. Nonoperative management was associated with a high rate of recurrent SBO: 31% after a median follow-up of 17 months. Of the patients managed operatively, 60% underwent immediate surgery and 40% underwent surgery after attempted nonoperative management. Patients in whom nonoperative management failed underwent surgery after a median of 2 days, and 89% underwent surgery within 5 days. The rate of bowel resection was high (29%) among those who underwent delayed surgery. Surgery after failed nonoperative management was associated with a mortality of 14% versus 3% for those who underwent immediate surgery; however, this difference was not significant., Conclusion: These data suggest that some elderly patients with SBO may be waiting too long for surgery.

Mitochondrial dysfunction is known to play a pivotal role in cell death mechanisms following traumatic brain injury (TBI). N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclosporin A (CsA) analog, inhibits the mitochondrial permeability transition pore (mPTP) and has been shown to be neuroprotective following TBI in mice. However, the translation of the neuroprotective effects of mPTP inhibitors, including CsA and NIM811, into improved cognitive end points has yet to be fully investigated. Therefore, to build upon these results, a severe unilateral controlled cortical impact model of TBI was used in the present study to establish a dose-response curve for NIM811 in rats. The findings demonstrate that the neuroprotection afforded by NIM811 is dose dependent, with the 10 mg/kg dose being the most effective dose. Once the dose response was established, we evaluated the effect of the optimal dose of NIM811 on behavior, mitochondrial bioenergetics, and mitochondrial oxidative damage following TBI. For behavioral studies, rats were administered NIM811 at 15 min and 24 h post-injury, with cognitive testing beginning 10 days post-injury. Mitochondrial studies involved a single injection of NIM811 at 15 min post-injury followed by mitochondrial isolation at 6 h post-injury. The results revealed that the optimal dose of NIM811 improves cognition, improves mitochondrial functioning, and reduces oxidative damage following TBI.

Traumatic injury to the mammalian spinal cord is a highly dynamic process characterized by a complex pattern of pervasive and destructive biochemical and pathophysiological events that limit the potential for functional recovery. Currently, there are no effective therapies for the treatment of spinal cord injury (SCI) and this is due, in part, to the widespread impact of the secondary injury cascades, including edema, ischemia, excitotoxicity, inflammation, oxidative damage, and activation of necrotic and apoptotic cell death signaling events. In addition, many of the signaling pathways associated with these cascades intersect and initiate other secondary injury events. Therefore, it can be argued that therapeutic strategies targeting a specific biochemical cascade may not provide the best approach for promoting functional recovery. A "systems approach" at the subcellular level may provide a better strategy for promoting cell survival and function and, as a consequence, improve functional outcomes following SCI. One such approach is to study the impact of SCI on the biology and function of mitochondria, which serve a major role in cellular bioenergetics, function, and survival. In this review, we will briefly describe the importance and unique properties of mitochondria in the spinal cord, and what is known about the response of mitochondria to SCI. We will also discuss a number of strategies with the potential to promote mitochondrial function following SCI.

Traumatic spinal cord injury (SCI) causes marked neuropathological changes in the spinal cord, resulting in limited functional recovery. Currently, there are no effective treatments, and the mechanisms underlying these neuropathological changes are not completely understood. In this study, two-dimensional gel electrophoresis coupled with mass spectrometry was used to investigate injury-related changes in the abundance (SYPRO Ruby stain) and phosphorylation (Pro-Q Diamond stain) of proteins from the soluble fraction of the lesion epicenter at 24 h following SCI. Over 1500 SYPRO Ruby-stained spots and 100 Pro-Q Diamond-stained spots were examined. We identified 26 unique proteins within 38 gel spots that differentially changed in abundance, phosphorylation, or both in response to SCI. Protein redundancies among the gel spots were likely due to differences in proteolysis, post-translational modifications, and the existence of isoforms. The proteins affected were blood-related proteins, heat-shock proteins, glycolytic enzymes, antioxidants, and proteins that function in cell structure, cell signaling, DNA damage, and protein degradation. These protein changes post injury may suggest additional avenues of investigation into the underlying molecular mechanisms responsible for the pathophysiological consequences of SCI.

The goal of the present study was to examine the neuroprotective and functional significance of targeting both N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity and oxidative stress using a dual-acting compound, Neu2000, in rat model of moderate spinal cord injury (SCI). An initial set of experiments was conducted in uninjured rats to study the pharmacokinetic profile of Neu2000 following intraperitoneal and intravenous administration. A second experiment measured free radical production in mitochondria isolated from sham or injured spinal cords of animals receiving vehicle or Neu2000 treatment. A third set of animals was divided into three treatment groups consisting of vehicle treatment, a single dose of Neu2000 (50 mg/kg) administered at 10 min following injury, or a repeated treatment paradigm consisting of a single bolus of Neu2000 at 10 min following injury (50 mg/kg) plus a maintenance dose (25 mg/kg) administered every 24 h for an additional 6 days. Animals were tested once a week for a period of 6 weeks for evidence of locomotor recovery in an open field and kinematic analysis of fine motor control using the DigiGait Image Analysis System. At the end of the testing period, spinal cord reconstruction was performed to obtain nonbiased stereological measures of tissue sparing. The results of this study demonstrate that Neu2000 treatment significantly reduced the production of mitochondrial free radicals and improved locomotor outcomes that were associated with a significant increase in the volume of spared spinal cord tissue.

Background: To determine if chronic elevation of the inflammatory cytokine, tumor necrosis factor-alpha (TNFalpha), will affect infarct volume or cortical perfusion after focal cerebral ischemia., Methods: Transgenic (TNFalpha-Tg) rats overexpressing the murine TNFalpha gene in brain were prepared by injection of mouse DNA into rat oocytes. Brain levels of TNFalpha mRNA and protein were measured and compared between TNFalpha-Tg and non-transgenic (non-Tg) littermates. Mean infarct volume was calculated 24 hours or 7 days after one hour of reversible middle cerebral artery occlusion (MCAO). Cortical perfusion was monitored by laser-Doppler flowmetry (LDF) during MCAO. Cortical vascular density was quantified by stereology. Post-ischemic cell death was assessed by immunohistochemistry and regional measurement of caspase-3 activity or DNA fragmentation. Unpaired t tests or analysis of variance with post hoc tests were used for comparison of group means., Results: In TNFalpha-Tg rat brain, the aggregate mouse and rat TNFalpha mRNA level was fourfold higher than in non-Tg littermates and the corresponding TNFalpha protein level was increased fivefold (p

Cyclosporin A (CsA) is a potent immunosuppressive drug shown to inhibit mitochondrial permeability transition (mPT). Although the therapeutic efficacy of CsA in traumatic brain injury is being investigated, CsA is highly neurotoxic and any neuroprotective effect in models of spinal cord injury (SCI) is unclear. NIM811 is a non-immunosuppressive CsA derivative that inhibits mPT, and is significantly less cytotoxic than CsA. Presently, we investigated the effects of NIM811 post-treatment on indices of apoptosis, lesion size, and tissue sparing at acute time-points following SCI. Adult rats received a "mild/moderate" contusion to the spinal cord, and were administered either 20 mg/kg NIM811 or vehicle by oral gavage 15 min later. One group of rats was euthanized at 1, 4, or 24 h post-injury, and the cytosolic levels of cytochrome c and fragmented DNA in the spinal cord were quantified. The remaining rats received an additional dose of NIM811 or vehicle at 24 h post-injury, and were euthanized on day 7 for morphometric assessments of the lesion and tissue spared. Control groups included rats that received sham surgery or no surgery. The results revealed that NIM811 post-treatment reduced the cytosolic levels of cytochrome c and fragmented DNA during the first 24 h following SCI. NIM811 also reduced the volume of the lesion, and enhanced the volumes of spared gray and white matter at 7 days post-injury. Together, these findings suggest that NIM811 treatment promoted tissue survival following SCI, in part, through inhibition of apoptotic mechanisms. This is the first study to demonstrate the therapeutic potential of NIM811 post-treatment in a model of acute SCI, and supports the need for continued investigation into NIM811 as a neuroprotective treatment for human SCI.

Trauma to the spinal cord causes a cascade of secondary events, such as mitochondrial dysfunction, which disrupts cellular functions and ultimately leads to cell death. Cyclosporin A (CsA) is a potent immunosuppressant that promotes mitochondrial function by inhibiting mitochondrial permeability transition (mPT). Clinical trials examining CsA in traumatic brain injury are currently under-way, but CsA is potentially neurotoxic. NIM811 is a non-immunosuppressive CsA derivative that inhibits mPT at nanomolar concentrations and with significantly less cytotoxicity than CsA. In the present study, we investigated the effects of NIM811 treatment on mitochondrial bioenergetics and the production of reactive oxygen species following spinal cord injury (SCI) in rats. Rats were pretreated with NIM811 or vehicle, and after 15 min the rats received a "mild/moderate" spinal cord contusion. After 24 h, the spinal cords were rapidly removed and synaptosomal mitochondria were isolated. NIM811 pretreatment significantly improved mitochondrial respiratory control ratios, and the maximal electron transport capacity of complex I and II, as well as their ATP-producing capacity. Consistent with the improvements in mitochondrial function, NIM811 pretreatment significantly decreased free radical production in isolated mitochondria. These studies are the first to demonstrate the therapeutic potential of CsA derivatives in a model of SCI, and support the need for continued investigation of compounds like NIM811 as an acute treatment for human SCI.

Injury to the spinal cord not only disrupts the functioning of spinal circuits at the site of the impact, but also limits sensorimotor function caudal to the level of the lesion. Ratings of gross locomotor skill are generally used to quantify locomotor recovery following spinal cord injury (SCI). The purpose of this study was to assess behavioral recovery following SCI with three tasks: (1) BBB ratings, (2) walking on a horizontal ladder, and (3) footprint analyses. Behavioral testing was conducted for 6 postoperative weeks, and then the spinal cords were processed for the amount of white matter spared. As expected, BBB ratings dramatically decreased and then improved during recovery. The number of hindlimb foot-faults on the horizontal ladder increased after injury and remained elevated during the recovery period. Footprint analyses revealed that sham-control rats used several different gaits to cross the runway. In contrast, the locomotor function of rats with a SCI was impaired throughout the postoperative period. Some locomotor parameters of the injured rats improved slightly (velocity, stride length, stride duration, stance duration), some did not change (interlimb coordination, swing duration, forelimb base of support, hindpaw angle), and others declined (hindlimb base of support) during the recovery period. Together, these results show that gross locomotor skill improved after SCI, while recovery of fine locomotor function was more limited. Multiple tests should be included in future experiments in order to assess gross and fine changes in sensorimotor function following SCI.

Spinal cord injury (SCI) initiates a cascade of biochemical changes that results in necrotic and apoptotic cell death. There is evidence that caspase-3 activation and apoptotic cell death occur within hours after SCI. However, the time course and cellular localization of activated caspase-3 has not been examined. Such information is essential because caspase-3-independent apoptotic pathways do exist. In this experiment, we describe the distribution of and cell types containing activated caspase-3 at 4 hr, 1 day, 2 days, 4 days, and 8 days following SCI in rats. Numerous caspase-3-positive cells were observed at 4 hr and 1 day postinjury and colocalized most often with CC1, a marker for oligodendroglia. Both markers disappeared near the injury epicenter over the next several days. Activated caspase-3 was again present in the injured spinal cord on postoperative day 8, which coincided with a reemergence of CC1-positive cells. Many of these CC1-positive cells again colocalized activated caspase-3. NeuN-positive neurons of the dorsal horn were occasionally immunopositive for activated caspase-3 at early time points. OX42-positive microglia/macrophages rarely contained activated caspase-3. The results indicate a biphasic pattern of caspase-3 activation during the first 8 days postinjury, suggesting that at least two mechanisms activate caspase-3 following SCI. This time-course study provides a framework for investigating and understanding the different signaling events contributing to this biphasic pattern of caspase-3 activation.

The purpose of this study was to investigate the potential neuroprotective efficacy of the mitochondrial uncoupler 2,4-dinitrophenol (DNP) in rats following a mild to moderate spinal cord contusion injury. Animals received intraperitoneal injections of vehicle (DMSO) or 5 mg/mL of DNP prior to injury. Twenty-four hours following surgery, mitochondrial function was assessed in mitochondria isolated from spinal cord synaptosomes. In addition, synaptosomes were used to measure indicators of reactive oxygen species formation, lipid peroxidation, and protein oxidation. Relative to vehicle-treated animals, pretreatment with DNP maintained mitochondrial bioenergetics and significantly decreased reactive oxygen species levels, lipid peroxidation, and protein carbonyl content following spinal cord injury. Furthermore, pretreatment with DNP significantly increased the amount of remaining white matter at the injury epicenter 6 weeks after injury. These results indicate that treatment with mitochondrial uncoupling agents may provide a novel approach for the treatment of secondary injury following spinal cord contusion.

It has long been recognized that much of the post-traumatic degeneration of the spinal cord following injury is caused by a multi-factorial secondary injury process that occurs during the first minutes, hours, and days after spinal cord injury (SCI). A key biochemical event in that process is reactive oxygen-induced lipid peroxidation (LP). In 1990 the results of the Second National Acute Spinal Cord Injury Study (NASCIS II) were published, which showed that the administration of a high-dose regimen of the glucocorticoid steroid methylprednisolone (MP), which had been previously shown to inhibit post-traumatic LP in animal models of SCI, could improve neurological recovery in spinal-cord-injured humans. This resulted in the registration of high-dose MP for acute SCI in several countries, although not in the U.S. Nevertheless, this treatment quickly became the standard of care for acute SCI since the drug was already on the U.S. market for many other indications. Subsequently, it was demonstrated that the non-glucocorticoid 21-aminosteroid tirilazad could duplicate the antioxidant neuroprotective efficacy of MP in SCI models, and evidence of human efficacy was obtained in a third NASCIS trial (NASCIS III). In recent years, the use of high-dose MP in acute SCI has become controversial largely on the basis of the risk of serious adverse effects versus what is perceived to be on average a modest neurological benefit. The opiate receptor antagonist naloxone was also tested in NASCIS II based upon the demonstration of its beneficial effects in SCI models. Although it did not a significant overall effect, some evidence of efficacy was seen in incomplete (i.e., paretic) patients. The monosialoganglioside GM1 has also been examined in a recently completed clinical trial in which the patients first received high-dose MP treatment. However, GM1 failed to show any evidence of a significant enhancement in the extent of neurological recovery over the level afforded by MP therapy alone. The present paper reviews the past development of MP, naloxone, tirilazad, and GM1 for acute SCI, the ongoing MP-SCI controversy, identifies the regulatory complications involved in future SCI drug development, and suggests some promising neuroprotective approaches that could either replace or be used in combination with high-dose MP.

Increased levels of free fatty acids and, in particular, arachidonic acid can lead to induction of apoptosis of spinal cord neurons. Because of the importance of neurotrophic factors in cell survival and death, mRNA and protein levels of brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (FGF-2) were studied in cultured spinal cord neurons treated with arachidonic acid. In addition, the present study focused on the effects of nicotine and neuronal nicotinic acetylcholine receptors (nAChRs) on these processes. A 2-h exposure to arachidonic acid markedly diminished expression of BDNF and FGF-2. These effects were fully prevented by pretreatment with 10 microM nicotine. Mecamylamine (a non-specific antagonist of nAChRs) and alpha-bungarotoxin (a specific antagonist of the nAChRalpha7) completely inhibited nicotine-mediated protection against arachidonic acid-induced alterations of BDNF and FGF-2. In addition, nicotine, BDNF and FGF-2 fully protected against arachidonic acid-induced apoptosis of spinal cord neurons. BDNF and FGF-2 were effective in prevention of apoptotic cell death even when applied 2 h after the beginning of arachidonic acid treatment. These results suggest that arachidonic acid can induce apoptosis of spinal cord neurons by depletion of neurotrophic factors and that nicotine can protect against these effects through the nAChRalpha7-mediated pathway.

The purpose of this study was to examine the effects of inhibiting ionotropic glutamate receptor subtypes on measures of oxidative stress events at acute times following traumatic spinal cord injury (SCI). Rats received a moderate contusion injury and 15 min later were treated with one of two doses of 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzol[f]quinoxaline-7-sulfonamide disodium (NBQX), MK-801, or the appropriate vehicle. At 4 h following injury, spinal cords were removed and a crude synaptosomal preparation obtained to examine mitochondrial function using the MTT assay, as well as measures of reactive oxygen species (ROS), lipid peroxidation, and glutamate and glucose uptake. We report here that intraspinal treatment with either 15 or 30 nmol of NBQX improves mitochondrial function and reduces the levels of ROS and lipid peroxidation products. In contrast, MK-801, given intravenously at doses of 1.0 or 5.0 mg/kg, was without effect on these same measures. Neither drug treatment had an effect on glutamate or glucose uptake, both of which are reduced at acute times following SCI. Previous studies have documented that drugs acting on non-N-methyl-D-aspartate (NMDA) receptors exhibit greater efficacy compared to NMDA receptor antagonists on recovery of function and tissue sparing following traumatic spinal cord injury. The results of this study provide a potential mechanism by which blockade of the non-NMDA ionotropic receptors exhibit positive effects following traumatic SCI.

An expanded polyglutamine domain in huntingtin underlies the pathogenic events in Huntington disease (HD), characterized by chorea, dementia and severe weight loss, culminating in death. Transglutaminase (TGase) may be critical in the pathogenesis, via cross-linking huntingtin. Administration of the TGase competitive inhibitor, cystamine, to transgenic mice expressing exon 1 of huntingtin containing an expanded polyglutamine repeat, altered the course of their HD-like disease. Cystamine given intraperitoneally entered brain where it inhibited TGase activity. When treatment began after the appearance of abnormal movements, cystamine extended survival, reduced associated tremor and abnormal movements and ameliorated weight loss. Treatment did not influence the appearance or frequency of neuronal nuclear inclusions. Unexpectedly, cystamine treatment increased transcription of one of the two genes shown to be neuroprotective for polyglutamine toxicity in Drosophila, dnaj (also known as HDJ1 and Hsp40 in humans and mice, respectively). Inhibition of TGase provides a new treatment strategy for HD and other polyglutamine diseases.

We report here that activation of the caspase-3 apoptotic cascade in spinal cord injury is regulated, in part, by calcineurin-mediated BAD dephosphorylation. BAD, a proapoptotic member of the bcl-2 gene family, is rapidly dephosphorylated after injury, dissociates from 14-3-3 in the cytosol, and translocates to the mitochondria of neurons where it binds to Bcl-x(L). Pretreatment of animals with FK506, a potent inhibitor of calcineurin activity, or MK801, an NMDA glutamate receptor antagonist, blocked BAD dephosphorylation and abolished activation of the caspase-3 apoptotic cascade. These findings extend previous in vitro observations and are the first to implicate the involvement of glutamate-mediated calcineurin activation and BAD dephosphorylation as upstream, premitochondrial signaling events leading to caspase-3 activation in traumatic spinal cord injury.

The potential use of riluzole (a glutamate release inhibitor) alone or in combination with methyl-prednisolone (MP) in treating acute spinal cored injury (SCI) was examined. Rats received a contusion injury to the spinal cord using the NYU impactor and were treated with vehicle, riluzole (8 mg/kg), MP(30 mg/kg), or riluzole + MP at 2 and 4 h following injury. Animals continued to receive riluzole treatment (8 mg/kg) for a period of 1 week. The animals were then tested weekly for functional recovery using the BBB open field locomotor score. At the end of testing (6 weeks after injury), each spinal cord was examined for the amount of remaining tissue at the injury site and a myelination index was used to quantify remaining axons in the ventromedial white matter. In this study, only the combination treatment was found to significantly improve behavioral recovery as assessed using the BBB open field locomotor scale. In addition, the combination treatment promoted tissue sparing at the lesion epicenter, but had no clear effect on the index of myelination. The results of this study clearly demonstrate the potential beneficial effects of a combination approach in the treatment of traumatic SCI.

Background & Aims: Hepatic osteodystrophy is a complication of primary biliary cirrhosis (PBC). Allelic polymorphisms of the vitamin D receptor (VDR) gene are related to bone mineral density (BMD) in normal cohorts and those with primary osteoporosis. We sought to establish the prevalence of reduced bone mass in PBC, correlate BMD with VDR gene polymorphisms, and identify risk factors for the development of hepatic osteodystrophy., Methods: Seventy-two female patients with PBC were evaluated prospectively. Clinical information, BMD assessment, disease severity, and osteoporosis risk factors were documented, and multivariate regression modeling was performed., Results: Twenty-four percent of the patients were osteoporotic at the lumbar spine and 32% at the femur. Severe bone loss (z score <-2.0) occurs 4 times more frequently in patients with PBC compared with controls. Body weight (P = 0.003) and postmenopausal status (P = 0.012) correlated independently with BMD. VDR genotype (P = 0.01) correlated with lower BMD at the spine only., Conclusions: Osteoporosis is a common complication of PBC. VDR genotype predicts lower BMD in patients with PBC. Studies are warranted to investigate the mechanism(s) by which VDR as well as other candidate genes may contribute to the development of hepatic osteodystrophy in PBC.

Traumatic spinal cord injury often results in complete loss of voluntary motor and sensory function below the site of injury. The long-term neurological deficits after spinal cord trauma may be due in part to widespread apoptosis of neurons and oligodendroglia in regions distant from and relatively unaffected by the initial injury. The caspase family of cysteine proteases regulates the execution of the mammalian apoptotic cell death program. Caspase-3 cleaves several essential downstream substrates involved in the expression of the apoptotic phenotype in vitro, including gelsolin, PAK2, fodrin, nuclear lamins and the inhibitory subunit of DNA fragmentation factor. Caspase-3 activation in vitro can be triggered by upstream events, leading to the release of cytochrome c from the mitochondria and the subsequent transactivation of procaspase-9 by Apaf-1. We report here that these upstream and downstream components of the caspase-3 apoptotic pathway are activated after traumatic spinal cord injury in rats, and occur early in neurons in the injury site and hours to days later in oligodendroglia adjacent to and distant from the injury site. Given these findings, targeting the upstream events of the caspase-3 cascade has therapeutic potential in the treatment of acute traumatic injury to the spinal cord.

The levels of nerve growth factor (NGF) mRNA can be regulated in vitro and in vivo in the hippocampal formation by events associated with pharmacological activation of glutamate receptors. In the present study, the level of NGF mRNA in the hippocampal formation was examined following an intrahippocampal injection of 1 nmole fluorocitrate, which temporarily inhibits the astrocyte metabolic activity in vivo. Consistent with previous findings, fluorocitrate treatment significantly increased glutamate levels and decreased glutamine levels in the dentate gyrus as determined by in vivo microdialysis. The increased ratio of glutamate to glutamine was followed by a significant increase in NGF mRNA expression selectively in dentate gyrus granule cells. The effects of increasing glutamate levels were blocked by pretreatment with 50 nmole 2-amino-5-phosphonovalerate (AP5), a competitive antagonist that acts at the N-methyl-D-aspartate (NMDA) glutamate receptor subtype. These findings suggest that NGF mRNA expression is regulated, in part, by changes in endogenous glutamate levels, partially through enhanced excitatory neurotransmission through NMDA receptors.

Radioactively labeled RNA probes in conjunction with in situ hybridization histochemistry have become a useful method for studying gene expression in the central nervous system. We used digoxigenin-labeled uridine triphosphate to synthesize cRNA probes for localization of nerve growth factor receptor (NGFR) mRNA in the rat basal forebrain. Detection of cells containing digoxigenin-labeled NGFR mRNA was accomplished using a digoxigenin antibody conjugated with alkaline phosphatase. NGFR mRNA-positive cells were distributed in three major cell groups in the basal forebrain: the medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. This technique provides a rapid and sensitive method for high-resolution detection of mRNA species in the central nervous system, as well as the potential for co-localization of two different mRNA species within individual cells.