The evolution of mammalian Rem2: unraveling the impact of purifying selection and coevolution on protein function, and implications for human disorders.

Rad And Gem-Like GTP-Binding Protein 2 (Rem2), a member of the RGK family of Ras-like GTPases, is implicated in Huntington's disease and Long QT Syndrome and is highly expressed in the brain and endocrine cells. We examine the evolutionary history of Rem2 identified in various mammalian species, focusing on the role of purifying selection and coevolution in shaping its sequence and protein structural constraints. Our analysis of Rem2 sequences across 175 mammalian species found evidence for strong purifying selection in 70% of non-invariant codon sites which is characteristic of essential proteins that play critical roles in biological processes and is consistent with Rem2's role in the regulation of neuronal development and function. We inferred epistatic effects in 50 pairs of codon sites in Rem2, some of which are predicted to have deleterious effects on human health. Additionally, we reconstructed the ancestral evolutionary history of mammalian Rem2 using protein structure prediction of extinct and extant sequences which revealed the dynamics of how substitutions that change the gene sequence of Rem2 can impact protein structure in variable regions while maintaining core functional mechanisms. By understanding the selective pressures, protein- and gene - interactions that have shaped the sequence and structure of the Rem2 protein, we gain a stronger understanding of its biological and functional constraints.

Association of Cerebrovascular Injury and Secondary Vascular Insult With Poor Outcomes After Gunshot Wound to the Head in a Large Civilian Population.

BACKGROUND AND OBJECTIVES: Cerebrovascular injury (CVI) after civilian gunshot wound to the head (GSWH) likely contributes to poor outcomes, but little supporting evidence exists. The purpose of this study was to determine whether intracranial CVI from GSWH and secondary vascular insult (stroke or rehemorrhage) were associated with poor outcomes in a large civilian population. METHODS: This was a single-institution, retrospective cohort study on patients admitted between January 2014 and July 2022 at a large, metropolitan, level-1 trauma center. Multivariate regression models and propensity score matching were used. RESULTS: A total of 512 civilian patients presented with GSWH, and a cohort of 172 (33.5%) met inclusion criteria, with 143 (83.1%) males and a mean (SD) age of 34.3 (±14.2) years. The incidence of intracranial CVI was 50.6% (87/172 patients), and that of secondary vascular insult was 32.2% (28/172 patients). Bifrontal trajectories (adjusted odds ratio [aOR] 13.11; 95% CI 2.45-70.25; P = .003) and the number of lobes traversed by the projectile (aOR 3.18; CI 1.77-5.71; P < .001) were associated with increased odds of resultant CVI. Patients with CVI suffered higher rate of mortality (34% vs 20%; odds ratio [OR] 2.1; CI 0.78-5.85; P = .015) and were less likely to achieve a good functional outcome with a Glasgow Outcome Score of 4-5 (34% vs 68%; OR 0.24; CI 0.1-0.6; P = .004) at follow-up. Furthermore, patients with CVI and resultant secondary vascular insult had even worse functional outcomes (Glasgow Outcome Score 4-5, 16.7% vs 39.0%; aOR 0.012; CI 0.001-0.169, P = .001). CONCLUSION: Intracranial CVI from GSWH and associated secondary vascular insult are associated with poor outcomes. Given the high prevalence and potentially reversible nature of these secondary injuries, early screening with vascular imaging and treatment of underlying CVI may prove to be critical to improve outcomes by reducing stroke and rehemorrhage incidence.

Complication rate of overlapping versus nonoverlapping functional and stereotactic surgery: a retrospective cohort study.

OBJECTIVE: Overlapping surgery, in which one attending surgeon manages two overlapping operating rooms (ORs) and is present for all the critical portions of each procedure, is an important policy that improves healthcare access for patients and case volumes for surgeons and surgical trainees. Despite several studies demonstrating the safety and efficacy of overlapping neurosurgical operations, the practice of overlapping surgery remains controversial. To date, there are no studies that have investigated long-term complication rates of overlapping functional and stereotactic neurosurgical procedures. The primary objective of this study was to investigate the 1-year complication rates and OR times for nonoverlapping versus overlapping functional procedures. The secondary objective was to gain insight into what types of complications are the most prevalent and test for differences between groups. METHODS: Seven hundred eighty-three functional neurosurgical cases were divided into two cohorts, nonoverlapping (n = 342) and overlapping (n = 441). The American Society of Anesthesiologists (ASA) scale score was used to compare the preoperative risk for both cohorts. A complication was defined as any surgically related reason that required readmission, reoperation, or an unplanned emergency department or clinic visit that required intervention. Complications were subdivided into infectious and noninfectious. Chi-square tests, independent-samples t-tests, and uni- and multivariable logistic regressions were used to determine significance. RESULTS: There were no significant differences in mean ASA scale score (2.7 ± 0.6 for both groups, p = 0.997) or overall complication rates (8.8% nonoverlapping vs 9.8% overlapping, p = 0.641) between the two cohorts. Infections accounted for the highest percentage of complications in both cohorts (46.6% vs 41.8%, p = 0.686). There were no statistically significant differences between mean in-room OR time (187.5 ± 141.7 minutes vs 197.1 ± 153.0 minutes, p = 0.373) or mean open-to-close time (112.2 ± 107.9 minutes vs 121.0 ± 123.1 minutes, p = 0.300) between nonoverlapping and overlapping cases. CONCLUSIONS: There was no increased risk of 1-year complications or increased OR time for overlapping functional and stereotactic neurosurgical procedures compared with nonoverlapping procedures.

Trans-Sulcal Parafascicular Port-Based Resection of a Subcortical Occipital High-Grade Glioma: 2-Dimensional Operative Video.

Minimally invasive trans-sulcal parafascicular port-based approaches can provide safe access to a wide variety of deep brain lesions.1,2 This surgical video illustrates the use of a minimally invasive port-based approach for resection of a subcortical right medial occipital lobe lesion in a 63-year-old woman who presented with seizures and bilateral left homonymous hemianopia. MRI showed a 2.5-cm contrast enhancing lesion with a necrotic center and significant T2 and fluid-attenuated inversion recovery signal consistent with vasogenic edema. Diffusion tensor imaging revealed compromise of the right optic radiations and edema extending through the right corticospinal tract. A minimally invasive trans-sulcal parafascicular port-based approach to the deep occipital lobe and periatrial region was performed under full exoscopic visualization. A gross total tumor resection was achieved, and histopathology revealed an IDH1-mutant, MGMT-methylated-positive glioblastoma. The patient's presentation, rationale, key surgical steps, and outcomes are discussed, and informed consent for surgery was obtained. The participants and any identifiable individuals consented to the publication of their image. Images at 3:50 used with permission from Nico Corporation. All rights reserved.

Double Fascicular Nerve Transfer to Musculocutaneous Branches for Restoration of Elbow Flexion in Brachial Plexus Injury.

Background Restriction of elbow flexion significantly limits upper extremity function following brachial plexus injuries. In recent years, the double fascicular nerve transfer procedure utilizing ulnar and median nerve transfer to musculocutaneous branches has shown promising functional outcomes. Objective To evaluate restoration of elbow flexion following a double fascicular transfer in patients with brachial plexus injuries and identify predictors of poor outcomes. Methods This retrospective review included 10 consecutive patients with brachial plexus injuries involving C5-C6 root avulsions who underwent the double nerve transfer procedure. The mean follow-up was 12 months and the primary outcome was assessment of elbow flexion with the use of the Medical Research Council (MRC) scale. Results This procedure achieved elbow flexion of MRC grade M3 or higher in 50% of our cohort. Time interval from injury to surgery showed a statistically significant inverse association with functional recovery (r = -0.73, p = 0.016). Patients who had the surgery within six months of the injury, demonstrated higher MRC grades during the follow-up (p = 0.048). There was no association between elbow flexion recovery and age, body mass index (BMI), gender, hypertension, diabetes or smoking status. Conclusions The double fascicular transfer to musculocutaneous may be a safe and effective treatment for restoration of elbow flexion. The procedure is associated with superior functional outcomes when performed within the first six months from the injury.

Combined Radial to Axillary and Spinal Accessory Nerve (SAN) to Suprascapular Nerve (SSN) Transfers May Confer Superior Shoulder Abduction Compared with Single SA to SSN Transfer.

BACKGROUND: The restoration of shoulder function after brachial plexus injury is a high priority. Shoulder abduction and stabilization can be achieved by nerve transfer procedures including spinal accessory nerve (SAN) to suprascapular nerve (SSN) and radial to axillary nerve transfer. The objective of this study is to compare functional outcomes after SAN to SSN transfer versus the combined radial to axillary and SA to SSN transfer. METHODS: This retrospective chart review included 14 consecutive patients with brachial plexus injury who underwent SAN to SSN transfer, 4 of whom had both SA to SSN and radial to axillary nerve transfer. RESULTS: SAN to SSN transfer achieved successful shoulder abduction (≥M3) in 64.3% of this cohort (9/14). During the long-term follow-up, patients achieved an average increase of 67.5° in shoulder abduction. There was no association between motor recovery and time from injury to surgery, age, body mass index (BMI), sex, or smoking status. The 4 patients who had SAN to SSN combined with radial to axillary nerve transfer demonstrated a statistically significant increase in the range of abduction (median, 90° vs. 42.5°, respectively; P = 0.022) compared with those who had SAN to SSN transfer alone; however, the difference in Medical Research Council (MRC) grades (MRC > M3) did not reach statistical significance (P = 0.07). CONCLUSIONS: Patients with brachial plexus injury and an intact C7 root could benefit from radial to axillary transfer in addition to SAN to SSN transfer. There was no association between recovery of shoulder abduction and time interval from injury to surgery, age, sex, smoking, and BMI.

Association of Overlapping Surgery With Patient Outcomes in a Large Series of Neurosurgical Cases.

Importance: Overlapping surgery (OS) is common. However, there is a dearth of evidence to support or refute the safety of this practice. Objective: To determine whether OS is associated with worsened morbidity and mortality in a large series of neurosurgical cases. Design, Setting, and Participants: A retrospective cohort study was completed for patients who underwent neurosurgical procedures at Emory University Hospital, a large academic referral hospital, between January 1, 2014, and December 31, 2015. Patients were operated on for pathologies across the spectrum of neurosurgical disorders. Propensity score weighting and logistic regression models were executed to compare outcomes for patients who received nonoverlapping surgery and OS. Investigators were blinded to study cohorts during data collection and analysis. Main Outcomes and Measures: The primary outcome measures were 90-day postoperative mortality, morbidity, and functional status. Results: In this cohort of 2275 patients who underwent neurosurgery, 1259 (55.3%) were female, and the mean (SD) age was 52.1 (16.4) years. A total of 972 surgeries (42.7%) were nonoverlapping while 1303 (57.3%) were overlapping. The distribution of American Society of Anesthesiologists score was similar between nonoverlapping surgery and OS cohorts. Median surgical times were significantly longer for patients in the OS cohort vs the nonoverlapping surgery cohort (in-room time, 219 vs 188 minutes; skin-to-skin time, 141 vs 113 minutes; both P < .001). Overlapping surgery was more frequently elective (93% vs 87%; P < .001). Regression analysis failed to demonstrate an association between OS and complications, such as mortality, morbidity, or worsened functional status. Measures of baseline severity of illness, such as admission to the intensive care unit and increased length of stay, were associated with mortality (intensive care unit: odds ratio [OR], 25.5; 95% CI, 6.22-104.67; length of stay: OR, 1.03; 95% CI, 1.00-1.05), morbidity (intensive care unit: OR, 1.85; 95% CI, 1.43-2.40; length of stay: OR, 1.06; 95% CI, 1.04-1.08), and unfavorable functional status (length of stay: OR, 1.03; 95% CI, 1.02-1.05). Conclusions and Relevance: These data suggest that OS can be safely performed if appropriate precautions and patient selection are followed. Data such as these will help determine health care policy to maximize patient safety.

Peripheral blood detection of systemic graft-specific xeno-antibodies following transplantation of human neural progenitor cells into the porcine spinal cord.

Extensive pre-clinical and clinical studies have searched for therapeutic efficacy of cell-based therapeutics in diseases of the Central Nervous System (CNS) with no other viable options. Allogeneic cells represent the primary source of these therapies and immunosuppressive regimens have been empirically employed based on experience with solid organ transplantation, attempting to avoid immune mediated graft rejection. In this study, we aimed to 1) characterize the host immune response to stem cells transplanted into the CNS and 2) develop a non-invasive method for detecting immune response to transplanted cell grafts. Human neural progenitor cells were transplanted into the spinal cord of 10 Göttingen minipigs, of which 5 received no immunosuppression and 5 received Tacrolimus. Peripheral blood samples were collected longitudinally for flow cytometry cross match studies. Necropsy was performed at day 21 and spinal cord tissue analysis. We observed a transient increase in xeno-reactive antibodies was detected on post-operative day 7 and 14 in pigs that did not receive immunosuppression. This response was not detected in pigs that received Tacrolimus immunosuppression. No difference in graft survival was observed between the groups. Infiltration of numerous immune mediators including granulocytes, T lymphocytes, and activated microglia, and complement deposition were detected. In summary, a systemic immunologic response to stem cell grafts was detected for two weeks after transplantation using peripheral blood. This could be used as a non-invasive biomarker by investigators for detection of immunologic rejection. However, the absence of a detectable response in peripheral blood does not rule out a parenchymal immune response.

Ferumoxytol Labeling of Human Neural Progenitor Cells for Diagnostic Cellular Tracking in the Porcine Spinal Cord with Magnetic Resonance Imaging.

We report on the diagnostic capability of magnetic resonance imaging (MRI)-based tracking of ferumoxytol-labeled human neural progenitor cells (hNPCs) transplanted into the porcine spinal cord. hNPCs prelabeled with two doses of ferumoxytol nanoparticles (hNPC-FLow and hNPC-FHigh ) were injected into the ventral horn of the spinal cord in healthy minipigs. Ferumoxytol-labeled grafts were tracked in vivo up to 105 days after transplantation with MRI. Injection accuracy was assessed in vivo at day 14 and was predictive of "on" or "off" target cell graft location assessed by histology. No difference in long-term cell survival, assessed by quantitative stereology, was observed among hNPC-FLow , hNPC-FHigh , or control grafts. Histological iron colocalized with MRI signal and engrafted human nuclei. Furthermore, the ferumoxytol-labeled cells retained nanoparticles and function in vivo. This approach represents an important leap forward toward facilitating translation of cell-tracking technologies to clinical trials by providing a method of assessing transplantation accuracy, delivered dose, and potentially cell survival. Stem Cells Translational Medicine 2017;6:139-150.

Magnetic Resonance Imaging-Guided Transplantation of Neural Stem Cells into the Porcine Spinal Cord.

BACKGROUND: Cell-based therapies are a promising treatment option for traumatic, tumorigenic and degenerative diseases of the spinal cord. Transplantation into the spinal cord is achieved with intravascular, intrathecal, or direct intraparenchymal injection. The current standard for direct injection is limited by surgical invasiveness, difficulty in reinjection, and the inability to directly target anatomical or pathological landmarks. The objective of this study was to present the proof of principle for minimally invasive, percutaneous transplantation of stem cells into the spinal cord parenchyma of live minipigs under MR guidance. METHODS: An MR-compatible spine injection platform was developed to work with the ClearPoint SmartFrame system (MRI Interventions Inc.). The system was attached to the spine of 2 live minipigs, a percutaneous injection cannula was advanced into the spinal cord under MR guidance, and cells were delivered to the cord. RESULTS: A graft of 2.5 × 106 human (n = 1) or porcine (n = 1) neural stem cells labeled with ferumoxytol nanoparticles was transplanted into the ventral horn of the spinal cord with MR guidance in 2 animals. Graft delivery was visualized with postprocedure MRI, and characteristic iron precipitates were identified in the spinal cord by Prussian blue histochemistry. Grafted stem cells were observed in the spinal cord of the pig injected with porcine neural stem cells. No postoperative morbidity was observed in either animal. CONCLUSION: This report supports the proof of principle for transplantation and visualization of pharmacological or biological agents into the spinal cord of a large animal under the guidance of MRI.

Preclinical Validation of Multilevel Intraparenchymal Stem Cell Therapy in the Porcine Spinal Cord.

BACKGROUND: Although multiple clinical trials are currently testing different stem cell therapies as treatment alternatives for many neurodegenerative diseases and spinal cord injury, the optimal injection parameters have not yet been defined. OBJECTIVE: To test the spinal cord's tolerance to increasing volumes and numbers of stem cell injections in the pig. METHODS: Twenty-seven female Göttingen minipigs received human neural progenitor cell injections using a stereotactic platform device. Cell transplantation in groups 1 to 5 (5-7 pigs in each) was undertaken with the intent of assessing the safety of an injection volume escalation (10, 25, and 50 µL) and an injection number escalation (20, 30, and 40 injections). Motor function and general morbidity were assessed for 21 days. Full necropsy was performed; spinal cords were analyzed for graft survival and microscopic tissue damage. RESULTS: No mortality or permanent surgical complications were observed during the 21-day study period. All animals returned to preoperative baseline within 14 days, showing complete motor function recovery. The histological analysis showed that there was no significant decrease in neuronal density between groups, and cell engraftment ranged from 12% to 31% depending on the injection paradigm. However, tissue damage was identified when injecting large volumes into the spinal cord (50 µL). CONCLUSION: This series supports the functional safety of various injection volumes and numbers in the spinal cord and gives critical insight into important safety thresholds. These results are relevant to all translational programs delivering cell therapeutics to the spinal cord.

Cellular therapeutics delivery to the spinal cord: technical considerations for clinical application.

Current literature demonstrates the efficacy of cell-based therapeutics in small animal models of varied spinal cord diseases. However, logistic challenges remain towards development of an optimized delivery approach to the human spinal cord. Clinical trials utilize a variety of methods to achieve this aim. In this article, the authors review currently employed delivery methods, compare the merits of alternate delivery paradigms, introduce their implementation in completed and ongoing clinical trials, and discuss promising near-term advances in image-guided delivery and in vivo graft tracking.

Surgical technique for spinal cord delivery of therapies: demonstration of procedure in gottingen minipigs.

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Stem cell therapy for the spinal cord.

Injury and disease of the spinal cord are generally met with a poor prognosis. This poor prognosis is due not only to the characteristics of the diseases but also to our poor ability to deliver therapeutics to the spinal cord. The spinal cord is extremely sensitive to direct manipulation, and delivery of therapeutics has proven a challenge for both scientists and physicians. Recent advances in stem cell technologies have opened up a new avenue for the treatment of spinal cord disease and injury. Stem cells have proven beneficial in rodent models of spinal cord disease and injury. In these animal models, stem cells have been shown to produce their effect by the dual action of cell replacement and the trophic support of the factors secreted by these cells. In this review we look at the main clinical trials involving stem cell transplant into the spinal cord, focusing on motor neuron diseases and spinal cord injury. We will also discuss the major hurdles in optimizing stem cell delivery methods into the spinal cord. We shall examine current techniques such as functional magnetic resonance imaging guidance and cell labeling and will look at the current research striving to improve these techniques. With all caveats and future research taken into account, this is a very exciting time for stem cell transplant into the spinal cord. We are only beginning to realize the huge potential of stem cells in a central nervous system setting to provide cell replacement and trophic support. Many more trials will need to be undertaken before we can fully exploit the attributes of stem cells.