Characterization of the glial scar tissue in a murine model of spinal cord compression, with focus on hyaluronan

Spinal cord injury (SCI) is a devastating neurological disorder that affects thousands of people each year. Although in recent decades significant progress has been made in relation to understanding the molecular and cellular events underlying the nervous damage, spinal cord injury is still a highly disabling condition for which there is no curative therapy. People affected by spinal cord injuries manifested dysfunction or loss, temporary or permanent, of motor, sensory and / or autonomic functions depending on the spinal lesion damaged. Currently, the incidence rate of this type of injury is approximately 15-40 cases per million people worldwide. At the origin of these lesions are: road accidents, falls, interpersonal violence and the practice of sports. In this work we placed the hypothesis that HA is one of the component of the scar tissue formed after a compressive SCI, that it is likely synthetised by the perilesional glial cells and that it might support the permeation of the glial scar during the late phase of SCI. Nowadays, much focus is drawn on the recovery of CNS function, made impossible after SCI due to the high content of sulfated proteoglycans in the extracellular matrix. Counterbalancing the ratio between these proteoglycans and hyaluronic acid could be one of the experimental therapy to re-permeate the glial scar tissue formed after SCI, making possible axonal regrowth and functional recovery. Therefore, we established a model of spinal cord compression in mice and studied the glial scar tissue, particularly through the characterization of the expression of enzymes related to the metabolism of HA and the subsequent concentration thereof at different distances of the lesion epicenter. Our results show that the lesion induced in mice shows results similar to those produced in human lesions, in terms of histologic similarities and behavioral results. but these animals demonstrate an impressive spontaneous reorganization mechanism of the spinal cord tissue that occurs after injury and allows for partial recovery of the functions of the CNS. As regards the study of the glial scar, changes were recorded at the level of mRNA expression of enzymes metabolizing HA i.e., after injury there was a decreased expression of HA synthases 1-2 (HAS 1-2) and an increase of the expression HAS3 synthase mRNA, as well as the enzymes responsible for the HA catabolism, HYAL 1-2. But the amount of HA measured through the ELISA test was found unchanged after injury, it is not possible to explain this fact only with the change of expression of enzymes. At two weeks and in response to SCI, we found synthesized HA by reactive astrocytes and probably by others like microglial cells as it was advanced by the HA/GFAP+ and HA/IBA1+ cells co-location.

Effects of hypoxic culture conditions on umbilical cord-derived human mesenchymal stem cells

Following cultivation of distinct mesenchymal stem cell (MSC) populations derived from human umbilical cord under hypoxic conditions (between 1.5% to 5% oxygen (O-2)) revealed a 2- to 3-fold reduced oxygen consumption rate as compared to the same cultures at normoxic oxygen levels (21% O-2). A simultaneous measurement of dissolved oxygen within the culture media from 4 different MSC donors ranged from 15 mu mol/L at 1.5% O-2 to 196 mu mol/L at normoxic 21% O-2. The proliferative capacity of the different hypoxic MSC populations was elevated as compared to the normoxic culture. This effect was paralleled by a significantly reduced cell damage or cell death under hypoxic conditions as evaluated by the cellular release of LDH whereby the measurement of caspase 3/7 activity revealed little if any differences in apoptotic cell death between the various cultures. The MSC culture under hypoxic conditions was associated with the induction of hypoxia-inducing factor-alpha (HIF-1 alpha) and an elevated expression of energy metabolism-associated genes including GLUT-1, LDH and PDK1. Concomitantly, a significantly enhanced glucose consumption and a corresponding lactate production could be observed in the hypoxic MSC cultures suggesting an altered metabolism of these human stem cells within the hypoxic environment.

Development of novel therapeutics and in vitro models for spinal cord injury

Spinal cord injury (SCI) is a devastating condition, which results from trauma to the cord, resulting in a primary injury response which leads to a secondary injury cascade, causing damage to both glial and neuronal cells. Following trauma, the central nervous system (CNS) fails to regenerate due to a plethora of both intrinsic and extrinsic factors. Unfortunately, these events lead to loss of both motor and sensory function and lifelong disability and care for sufferers of SCI. There have been tremendous advancements made in our understanding of the mechanisms behind axonal regeneration and remyelination of the damaged cord. These have provided many promising therapeutic targets. However, very few have made it to clinical application, which could potentially be due to inadequate understanding of compound mechanism of action and reliance on poor SCI models. This thesis describes the use of an established neural cell co-culture model of SCI as a medium throughput screen for compounds with potential therapeutic properties. A number of compounds were screened which resulted in a family of compounds, modified heparins, being taken forward for more intense investigation. Modified heparins (mHeps) are made up of the core heparin disaccharide unit with variable sulphation groups on the iduronic acid and glucosamine residues; 2-O-sulphate (C2), 6-O-sulphate (C6) and N-sulphate (N). 2-O-sulphated (mHep6) and N-sulphated (mHep7) heparin isomers were shown to promote both neurite outgrowth and myelination in the SCI model. It was found that both mHeps decreased oligodendrocyte precursor cell (OPC) proliferation and increased oligodendrocyte (OL) number adjacent to the lesion. However, there is a difference in the direct effects on the OL from each of the mHeps; mHep6 increased myelin internode length and mHep7 increased the overall cell size. It was further elucidated that these isoforms interact with and mediate both Wnt and FGF signalling. In OPC monoculture experiments FGF2 treated OPCs displayed increased proliferation but this effect was removed when co-treated with the mHeps. Therefore, suggesting that the mHeps interact with the ligand and inhibit FGF2 signalling. Additionally, it was shown that both mHeps could be partially mediating their effects through the Wnt pathway. mHep effects on both myelination and neurite outgrowth were removed when co-treated with a Wnt signalling inhibitor, suggesting cell signalling mediation by ligand immobilisation and signalling activation as a mechanistic action for the mHeps. However, the initial methods employed in this thesis were not sufficient to provide a more detailed study into the effects the mHeps have on neurite outgrowth. This led to the design and development of a novel microfluidic device (MFD), which provides a platform to study of axonal injury. This novel device is a three chamber device with two chambers converging onto a central open access chamber. This design allows axons from two points of origin to enter a chamber which can be subjected to injury, thus providing a platform in which targeted axonal injury and the regenerative capacity of a compound study can be performed. In conclusion, this thesis contributes to and advances the study of SCI in two ways; 1) identification and investigation of a novel set of compounds with potential therapeutic potential i.e. desulphated modified heparins. These compounds have multiple therapeutic properties and could revolutionise both the understanding of the basic pathological mechanisms underlying SCI but also be a powered therapeutic option. 2) Development of a novel microfluidic device to study in greater detail axonal biology, specifically, targeted axonal injury and treatment, providing a more representative model of SCI than standard in vitro models. Therefore, the MFD could lead to advancements and the identification of factors and compounds relating to axonal regeneration.

Spatiotemporal neuromodulation therapies engaging muscle synergies improve motor control after spinal cord injury.

Electrical neuromodulation of lumbar segments improves motor control after spinal cord injury in animal models and humans. However, the physiological principles underlying the effect of this intervention remain poorly understood, which has limited the therapeutic approach to continuous stimulation applied to restricted spinal cord locations. Here we developed stimulation protocols that reproduce the natural dynamics of motoneuron activation during locomotion. For this, we computed the spatiotemporal activation pattern of muscle synergies during locomotion in healthy rats. Computer simulations identified optimal electrode locations to target each synergy through the recruitment of proprioceptive feedback circuits. This framework steered the design of spatially selective spinal implants and real-time control software that modulate extensor and flexor synergies with precise temporal resolution. Spatiotemporal neuromodulation therapies improved gait quality, weight-bearing capacity, endurance and skilled locomotion in several rodent models of spinal cord injury. These new concepts are directly translatable to strategies to improve motor control in humans.

Sex cord-stromal tumours of the ovary: a comprehensive update review

Ovarian sex cord-stromal tumors are infrequent and represent approximately 7% of all primary ovarian tumors. This histopathologic ovarian tumor group differs considerably from the more prevalent epithelial ovarian tumors. Although sex cord-stromal tumors present in a broad age group, the majority tend to present as a low-grade disease that usually follows a nonaggressive clinical course in younger patients. Furthermore, because the constituent cells of these tumors are engaged in ovarian steroid hormone production (e.g., androgens, estrogens, and corticoids), sex cord-stromal tumors are commonly associated with various hormone-mediated syndromes and exhibit a wide spectrum of clinical features ranging from hyperandrogenic virilizing states to hyperestrogenic manifestations. The World Health Organization sex cord-stromal tumor classification has recently been revised, and currently these tumors have been regrouped into the following clinicopathologic entities: pure stromal tumors, pure sex cord tumors, and mixed sex cord-stromal tumors. Moreover, some entities considered in the former classification (e.g., stromal luteoma, stromal tumor with minor sex cord elements, and gynandroblastoma) are no longer considered separate tumors in the current classification. Herein, we discuss and revise the ultrasonography, computed tomography, and magnetic resonance imaging characteristics of the different histopathologic types and clinicopathologic features of sex cord-stromal tumors to allow radiologists to narrow the differential diagnosis when facing ovarian tumors.

Short Term Outcome of Neonates Born With Abnormal Umbilical Cord Arterial Blood Gases

Background: Umbilical arterial blood gas (UABG) analysis is more objective than other methods for predicting neonatal outcome. Acidemic neonates may be at risk for unfavorable outcome after birth, but all neonates with abnormal arterial blood gas (ABG) analysis do not always have poor outcome. Objectives: This study was carried out to determine the short term outcome of the neonates born with an abnormal ABG. Patients and Methods: In a cohort prospective study 120 high risk mother-neonate pairs were enrolled and UABG was taken immediately after birth. All neonates with an umbilical cord pH less than 7.2 were considered as case group and more than 7.2 as controls. Outcomes like need to resuscitation, admission to newborn services and/or NICU), seizure occurrence, hypoxic ischemic encephalopathy (HIE), delayed initiation of oral feeding and length of hospital stay were recorded and compared between the two groups. P value less than 0.05 was considered as being significant. Results: Comparison of short term outcomes between normal and abnormal ABG groups were as the fallowing: need for advanced resuscitation 4 vs. 0 (P = 0.001), NICU admission 16 vs. 4 (P = 0.001), convulsion 2 vs. 0 (P = 0.496), HIE 17 vs. 4 (P = 0.002), delay to start oral feeding 16 vs. 4 (P = 0.001), mean hospital stay 4 vs. 3 days (P = 0.001). None of the neonates died in study groups. Conclusions: An umbilical cord PH less than 7.2 immediately after birth can be used as a prognostic factor for unfavorable short term outcome in newborns.

Cord Blood Alkaline Phosphatase as an Indicator of Neonatal Jaundice

Background: Management of hyperbilirubinemia remains a challenge for neonatal medicine because of the risk of neurological complications related to the toxicity of severe hyperbilirubinemia. Objectives: The purpose of this study was to examine the validity of cord blood alkaline phosphatase level for predicting neonatal hyperbilirubinemia. Patients and Methods: Between October and December 2013 a total of 102 healthy term infants born to healthy mothers were studied. Cord blood samples were collected for measurement of alkaline Phosphatase levels immediately after birth. Neonates were followed-up for the emergence of jaundice. Newborns with clinical jaundice were recalled and serum bilirubin levels measured. Appropriate treatment based on serum bilirubin level was performed. Alkaline phosphatase levels between the non-jaundiced and jaundiced treated neonates were compared. Results: The incidence of severe jaundice that required treatment among followed-up neonates was 9.8%. The mean alkaline phosphatase level was 309.09 ± 82.51 IU/L in the non-jaundiced group and 367.80 ± 73.82 IU/L in the severely jaundiced group (P = 0.040). The cutoff value of 314 IU/L was associated with sensitivity 80% and specificity 63% for predicting neonatal hyperbilirubinemia requiring treatment. Conclusions: The cord blood alkaline phosphatase level can be used as a predictor of severe neonatal jaundice.

Dipsacus asperoides (Xue Duan) inhibits spinal cord injury-induced inflammatory responses in rats

Purpose: To investigate the effect of Dipsacus asperoides (Xue Duan), a traditional Chinese medicine, on rats with spinal cord injury (SCI). Methods: In this study a total of 40 adult rats were used after inducing SCI where Xue Duan was applied on experimental group and phosphate-buffered saline (PBS) was administered in corresponding control groups. Intraperitoneal administration of both compounds for a period of four weeks (28 days) was carried out at a dose of 10 mg/kg/day. Bright field microscopy was performed on the tissues. Results: Bright Field microscopy of tissue sections showed significant reduction in cavity area that resulted from injury, that is from 0.19 ± 0.05 mm2 to 0.09 ± 0.03 mm2 (p < 0.01) in untreated and treated groups respectively. Similarly western blotting results showed a decrease in the expression of NF-kB p65 and I-kBα (p < 0.01). These two compounds are important in increasing secondary pathophysiology in SCI. The results for MPO activity also revealed significantly reduced infiltration of leukocytes to the injury site (p < 0.01). Conclusion: This study reveals the positive effect of the plant material in reducing inflammation in rats with traumatic SCI.

Dose-dependent protection of reseveratrol against spinal cord ischemic-reperfusion injury in rats

Purpose: To examine the protective effects of resveratrol (RESV) against spinal cord ischemic reperfusion (SCIR) injury. Methods: Forty-eight male rats were divided into six groups: sham-operated (control-I), SCIR-treated (SCIR-II), rats receiving 20 mg/kg of RESV with SCIR (RESV 20+SCIR-III), rats receiving 40 mg/kg of RESV with SCIR (RESV 40+SCIR-IV), rats receiving 60 mg/kg of RESV with SCIR (RESV 60+SCIR-V), and rats receiving 50 mg/kg of methylprednisolone (MP) with SCIR (MP + SCIR-VI), for 7 days prior to IR (pre-treatment) and 7 days after IR (post-treatment). Results: The levels of oxidative markers (TBARS, MPO) and inflammatory markers (IL-1β, IL-6, TNF-α, and NF-p65) were concomitantly suppressed in RESV-treated rats, which showed improved locomotor function. A pronounced increase in the activities of antioxidant enzymes (SOD, CAT and GSH) was noted in the RESV group compared with the MP and SCIR groups. RESV and MP supplementation increased neuronal count with decreased nuclear degeneration. RESV (40 mg) exhibited greater protective effect than 20 mg and 60 mg of RESV and 50 mg of MP. Conclusion: The results show the neurotherapeutic potential of RESV (40 mg) to attenuate oxidative stress and the inflammatory response to SCIR injury.

A preclinical study of spinal cord injury focused on cellular and molecular modifications as potential targets for innovative therapies

Spinal Cord Injury (SCI) is a devastating condition for human and animal health. In SCI particularly, neurons, oligodendrocytes precursor cells, and mature oligodendrocytes are highly vulnerable to the toxic microenvironment after the lesion and susceptible to the elevated levels of noxious stimuli. Thus the regenerative response of the organism in case of SCI is significantly reduced, and only little spontaneous amelioration is observed in lesioned patients during the early phases. This work mainly focuses on studying and characterizing the modification induced by the SCI in a preclinical animal model. We investigated the ECM composition in the spinal cord segments surrounding the primary lesion site at a gene expression level. We found Timp1 and CD44 as a crucial hub in the secondary cascade of SCI in both spinal cord segments surrounding the lesion site. Interestingly, a temporal and anatomical difference in gene expression, indicating a complex regulation of ECM genes after SCI that could be used as a tool for regenerative medicine. We also investigated the modification in synaptic plasticity-related gene expression in spinal and supraspinal areas involved in motor control. We confirmed the anatomical and temporal difference in gene expression in spinal cord tissue. This analysis suggests that a molecular mapping of the lesion-induced modification could be a useful tool for regenerative medicine. In the last part, we evaluated the efficacy of an implantable biopolymer loaded with an anti-inflammatory drug and a pro-myelinating agent on the acute phase of SCI in our preclinical model. We found a consistent reduction of the inflammatory state in the spinal lesion site and the cord's surrounding segments. Moreover, we found increased preservation of the spinal cord tissue with a related upregulation of neuronal and oligodendroglial markers after lesion. Our treatment showed effective ameliorating functional outcome and reducing the lesion extension in the chronic phase.

Late onset spinal cord ischemia after thoracoabdominal aortic aneurysm open repair

The aim of this study is to evaluate if spinal cord ischemia (SCI), especially its late presentation, and can be correlated to the results of intraoperative evoked potential monitoring (IOM). Methods. This study is a physician-initiated, retrospective, single-center, non-randomized study. Data from all patients undergoing a thoracoabdominal aortic aneurysm surgical repair (TAAA SR) between January 2016 and March 2020 IOM was collected and analyzed. Results. During the study period, 261 patients underwent TAAA SR with MEP/SSEPs monitoring [190 males, 73%; median age 65 (57-71)]. Thirty-seven patients suffered from SCI, for an overall rate of 14% (permanent 9%). When stratifying patients according to the SCI onset, 18 patients presented with an early (11 permanent) and 19 with a late SCI (<24h) (11 permanent). Of 261 patients undergoing TAAA SR with IOM, 15 were excluded due to changes in the upper extremity motor evoked potentials. For the remaining 246, the association between SCI and IOM was investigated: only irreversible IOM loss without peripheral changes have been found to be a risk factor for late onset SCI (p=.006). Furthermore, given that no statistical differences were found between the two groups when no IOM changes were recorded (p=.679), this situation cannot reliably rule out any SCI in our cohort. Independent risk factors for late spinal cord ischemia onset found at multivariate analysis were smoking history (p=.008), BMI>28 (p=.048) and TAAA extent II (p=.009). The irreversible MEP change without peripheral showed a trend of significance (p=.052). Conclusions. Evoked potential intraoperative monitoring is an important adjunct during thoracoabdominal aortic open repair to predict and possibly prevent spinal cord ischemia. Irreversible IOM loss without peripheral changes was predictive of late SCI, therefore more attention should be paid to the postoperative management of this subgroup of patients.

Influence Of Delivery Method On Neuroprotection By Bone Marrow Mononuclear Cell Therapy Following Ventral Root Reimplantation With Fibrin Sealant.

The present work compared the local injection of mononuclear cells to the spinal cord lateral funiculus with the alternative approach of local delivery with fibrin sealant after ventral root avulsion (VRA) and reimplantation. For that, female adult Lewis rats were divided into the following groups: avulsion only, reimplantation with fibrin sealant; root repair with fibrin sealant associated with mononuclear cells; and repair with fibrin sealant and injected mononuclear cells. Cell therapy resulted in greater survival of spinal motoneurons up to four weeks post-surgery, especially when mononuclear cells were added to the fibrin glue. Injection of mononuclear cells to the lateral funiculus yield similar results to the reimplantation alone. Additionally, mononuclear cells added to the fibrin glue increased neurotrophic factor gene transcript levels in the spinal cord ventral horn. Regarding the motor recovery, evaluated by the functional peroneal index, as well as the paw print pressure, cell treated rats performed equally well as compared to reimplanted only animals, and significantly better than the avulsion only subjects. The results herein demonstrate that mononuclear cells therapy is neuroprotective by increasing levels of brain derived neurotrophic factor (BDNF) and glial derived neurotrophic factor (GDNF). Moreover, the use of fibrin sealant mononuclear cells delivery approach gave the best and more long lasting results.

Cervical Injuries Scored According To The Subaxial Injury Classification System: An Analysis Of The Literature.

The Subaxial Injury Classification (SLIC) system and severity score has been developed to help surgeons in the decision-making process of treatment of subaxial cervical spine injuries. A detailed description of all potential scored injures of the SLIC is lacking. We performed a systematic review in the PubMed database from 2007 to 2014 to describe the relationship between the scored injuries in the SLIC and their eventual treatment according to the system score. Patients with an SLIC of 1-3 points (conservative treatment) are neurologically intact with the spinous process, laminar or small facet fractures. Patients with compression and burst fractures who are neurologically intact are also treated nonsurgically. Patients with an SLIC of 4 points may have an incomplete spinal cord injury such as a central cord syndrome, compression injuries with incomplete neurologic deficits and burst fractures with complete neurologic deficits. SLIC of 5-10 points includes distraction and rotational injuries, traumatic disc herniation in the setting of a neurological deficit and burst fractures with an incomplete neurologic deficit. The SLIC injury severity score can help surgeons guide fracture treatment. Knowledge of the potential scored injures and their relationships with the SLIC are of paramount importance for spine surgeons who treated subaxial cervical spine injuries.

Impact Of Pregabalin Treatment On Synaptic Plasticity And Glial Reactivity During The Course Of Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease that affects young adults. It is characterized by generating a chronic demyelinating autoimmune inflammation in the central nervous system. An experimental model for studying MS is the experimental autoimmune encephalomyelitis (EAE), induced by immunization with antigenic proteins from myelin. The present study investigated the evolution of EAE in pregabalin treated animals up to the remission phase. The results demonstrated a delay in the onset of the disease with statistical differences at the 10th and the 16th day after immunization. Additionally, the walking track test (CatWalk) was used to evaluate different parameters related to motor function. Although no difference between groups was obtained for the foot print pressure, the regularity index was improved post treatment, indicating a better motor coordination. The immunohistochemical analysis of putative synapse preservation and glial reactivity revealed that pregabalin treatment improved the overall morphology of the spinal cord. A preservation of circuits was depicted and the glial reaction was downregulated during the course of the disease. qRT-PCR data did not show immunomodulatory effects of pregabalin, indicating that the positive effects were restricted to the CNS environment. Overall, the present data indicate that pregabalin is efficient for reducing the seriousness of EAE, delaying its course as well as reducing synaptic loss and astroglial reaction.

Synaptic Rearrangement Following Axonal Injury: Old And New Players.

Following axotomy, the contact between motoneurons and muscle fibers is disrupted, triggering a retrograde reaction at the neuron cell body within the spinal cord. Together with chromatolysis, a hallmark of such response to injury is the elimination of presynaptic terminals apposing to the soma and proximal dendrites of the injured neuron. Excitatory inputs are preferentially eliminated, leaving the cells under an inhibitory influence during the repair process. This is particularly important to avoid glutamate excitotoxicity. Such shift from transmission to a regeneration state is also reflected by deep metabolic changes, seen by the regulation of several genes related to cell survival and axonal growth. It is unclear, however, how exactly synaptic stripping occurs, but there is substantial evidence that glial cells play an active role in this process. In one hand, immune molecules, such as the major histocompatibility complex (MHC) class I, members of the complement family and Toll-like receptors are actively involved in the elimination/reapposition of presynaptic boutons. On the other hand, plastic changes that involve sprouting might be negatively regulated by extracellular matrix proteins such as Nogo-A, MAG and scar-related chondroitin sulfate proteoglycans. Also, neurotrophins, stem cells, physical exercise and several drugs seem to improve synaptic stability, leading to functional recovery after lesion.