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.

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.

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.

Schistosomal myeloradiculopathy in a low-prevalence area: 27 cases (14 autochthonous) in Campinas, São Paulo, Brazil

Schistosomal myeloradiculopathy (SMR) is a form of schistosomiasis that is not linked with a high worm burden but rather is found in patients who have been sporadically exposed to Schistosoma mansoni. This paper aims to determine the occurrence of SMR in a low-endemic area with urban transmission in Campinas, São Paulo, Brazil. A retrospective study was performed, identifying confirmed cases in the two largest public hospitals on the region. Patients were diagnosed with SMR using standardised criteria, common clinical parameters, evidence of schistosomal infection and exclusion of other causes of myelopathy. A total of 27 patients were identified; 19 (85.2%) were men and four (14.8%) were women, ranging from 13-57 years of age (mean = 31.2; standard deviation = 12.8). Patients were classified as autochthonous (n = 14; 51.9%) or allochthonous (n = 11; 40.7%) and epidemiological data could not be obtained for two patients (7.4%). The clinical parameters of these patients were not different from previous studies. The sensitivity of serum immune reactions, cerebrospinal fluid immune reactions and parasitological stool examinations in identifying infected individuals was 87.5%, 93.8% and 40%, respectively. The epidemiological importance of these findings and their relationship with the control policies of schistosomiasis are discussed.

Nutrición enteral en el paciente neurológico: ¿es suficiente el contenido en vitamina D en las fórmulas de uso habitual?

INTRODUCTION Vitamin D deficiency produces inadequate bone mineralization, proximal muscle weakness, abnormal gait and increased risk of falls and fractures. Moreover, in epidemiological studies, has been associated with increased risk of cancer, autoimmune diseases, type 1 and 2 diabetes, rheumatoid arthritis, multiple sclerosis, infectious diseases, cardiovascular diseases and depression. When synthesis through the skin by sun exposure is not possible and the patient can not eat by mouth, as in the advanced stages of various neurological diseases, the supply of vitamin D has to be done by enteral nutrition. OBJECTIVES The aim of this study is to review the role of vitamin D in a common group of neurological conditions that often require artificial nutrition and analyze whether the vitamin D of different enteral nutrition formulas is adequate to meet the needs of this group of patients. RESULTS Numerous studies have shown the association between vitamin D deficiency and increased incidence of dementia, stroke and other neurodegenerative diseases. Interventions aimed to increase levels of vit. D and its effects on functional (falls, pain, quality of life) and cardiovascular goals (cardiovascular death, stroke, myocardial infarction, cardiovascular risk factors) have obtained as highlight data a clear reduction of falls and fractures, while the evidence for the other parameters studied is still limited and inconsistent. The content of calcium and vitamin D of enteral formulas is legislated in our country. The total amount of vitamin D for a daily intake of 1,500-2,000 kcal ranges between 300 and 1,600 IU/d (mean ± SD: 32.9 ± 8.5 mg/100 kcal) in the complete formulas for enteral nutrition most commonly used. 50% of the diets studied, for an intake of 2,000 kcal/d, and 90% for an intake of 1,500 kcal/d, provide less than 600 IU/d of vitamin D. DISCUSSION Some revised recently guidelines published recommendations of daily intake of vitamin D. The document published by the U.S. Institute of Medicine recommended for adults between 19 and 70 years, 600 IU/d and up from 70, proposes 800 IU/d of vitamin D. These amounts are deemed insufficient by other scientific societies to state that to achieve blood levels of 25 (OH) D equal or greater than 30 ng/ml may be required a daily intake of 1,500-2,000 IU and a number two or three times higher if previous deficiency exists. CONCLUSIONS Further controlled studies are needed to ascertain which is the appropriate dose of vitamin D in advanced stages of neurological disease, where sun exposure is difficult and unlikely. We suggest that the vitamin D content should probably be reconsidered in enteral nutrition formulas, which, in light of recent publications appear as clearly insufficient for standard energy intakes (1,500-2,000 kcal).

Cardiovascular and respiratory alterations in dogs during cervical myelography using ioversol

Subarachnoid infusion of most contrast mediums and the steps involved in performing a cervical myelography have adverse affects that can discourage its use in the radiographic diagnosis of spinal cord diseases. Thus, the cardiovascular and respiratory alterations associated with neck flexion, subarachnoid puncture, and cerebrospinal fluid drainage during subarachnoid infusion of ioversol (320mgI/mL) in dogs under general anesthesia using isoflurane were evaluated. The dogs received subarachnoid infusion of autologous cerebrospinal fluid kept at 38°C - control group (GC); ioversol 0.3mL/kg at 25°C (GI25) and ioversol 0.3mL/kg heated to 38°C (GI38). Each dog had its heart rate (HR), systolic arterial pressure (SAP), diastolic arterial pressure (DAP), respiratory rate (RR), oxyhemoglobin saturation (SaO2) and electrocardiography readings (PR and QT intervals) recorded. Group comparisons showed no statistical difference regarding neck positioning, subarachnoid puncture, and subarachnoid infusion of contrast medium on HR, RR and SaO2, cardiac rhythm or conduction. However, isoflurane significantly increased PR and QT intervals. Based on these findings, it is concluded that the steps involved in cervical myelography and the use of ioversol 320mgI/mL at 0.3mL/kg (25ºC and 38ºC) during cervical myelography did not result in relevant cardiovascular and respiratory alterations, except for an elevation in arterial pressure after injection of ioversol.

Estudo mielográfico comparativo entre meios de contraste iopamidol e ioexol em bezerros

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Fatores prognósticos associados ao tratamento cirúrgico da mielorradiculopatia espondilótica cervical

OBJETIVO: Identificar os fatores clínicos dos indivíduos, fatores sociais, ambientais e dos exames de imagem que se correlacionam ao resultado final de melhora neurológica em pacientes submetidos ao tratamento cirúrgico da mielopatia espondilótica cervical. MÉTODOS: A avaliação clínica foi quantificada pela escala deficitária da JOA. Analisamos 200 casos de mielorradiculopatia cervical, operados no HC-FMUSP, no período de janeiro de 1993 a janeiro de 2007. A média de segmento foi de 06 anos e 08 meses. A análise radiológica foi baseada nos critérios de instabilidade de White e scala de Kellgren. RESULTADOS: Em 80% houve melhora, 14% estabilização e em 6% piora do quadro neurológico. A piora neurológica não foi associada com nenhum fator clínico, ambiental ou de imagem. A melhora neurológica foi diretamente proporcional a menor idade na cirurgia, ausência de co-morbidade, sinal de Hoffman, atrofia muscular, hipersinal medular na RNM, menor período de evolução pré-operatório, melhor status neurológico pré-operatório e inversamente proporcional ao diâmetro AP do canal medular e multiplicidade de compressões. Identificou-se associação com o tabagismo. Mais de 70 anos, evolução superior a 24 meses, atrofia muscular, pontuação JOA igual ou inferior a sete pontos e diâmetro AP do canal inferior ou igual a seis mm não foram associado à melhora.

Management of faecal incontinence and constipation in adults with central neurological diseases.

BACKGROUND: People with neurological disease have a much higher risk of both faecal incontinence and constipation than the general population. There is often a fine line between the two conditions, with any management intended to ameliorate one risking precipitating the other. Bowel problems are observed to be the cause of much anxiety and may reduce quality of life in these people. Current bowel management is largely empirical with a limited research base. OBJECTIVES: To determine the effects of management strategies for faecal incontinence and constipation in people with neurological diseases affecting the central nervous system. SEARCH STRATEGY: We searched the Cochrane Incontinence Group Specialised Trials Register (searched 26 January 2005), the Cochrane Central Register of Controlled Trials (Issue 2, 2005), MEDLINE (January 1966 to May 2005), EMBASE (January 1998 to May 2005) and all reference lists of relevant articles. SELECTION CRITERIA: All randomised or quasi-randomised trials evaluating any types of conservative or surgical measure for the management of faecal incontinence and constipation in people with neurological diseases were selected. Specific therapies for the treatment of neurological diseases that indirectly affect bowel dysfunction were also considered. DATA COLLECTION AND ANALYSIS: Two reviewers assessed the methodological quality of eligible trials and two reviewers independently extracted data from included trials using a range of pre-specified outcome measures. MAIN RESULTS: Ten trials were identified by the search strategy, most were small and of poor quality. Oral medications for constipation were the subject of four trials. Cisapride does not seem to have clinically useful effects in people with spinal cord injuries (three trials). Psyllium was associated with increased stool frequency in people with Parkinson's disease but did not alter colonic transit time (one trial). Prucalopride, an enterokinetic did not demonstrate obvious benefits in this patient group (one study). Some rectal preparations to initiate defaecation produced faster results than others (one trial). Different time schedules for administration of rectal medication may produce different bowel responses (one trial). Mechanical evacuation may be more effective than oral or rectal medication (one trial). There appears to be a benefit to patients in one-off educational interventions from nurses. The clinical significance of any of these results is difficult to interpret. AUTHORS' CONCLUSIONS: There is still remarkably little research on this common and, to patients, very significant condition. It is not possible to draw any recommendation for bowel care in people with neurological diseases from the trials included in this review. Bowel management for these people must remain empirical until well-designed controlled trials with adequate numbers and clinically relevant outcome measures become available.

Management of faecal incontinence and constipation in adults with central neurological diseases.

BACKGROUND: People with neurological disease have a much higher risk of both faecal incontinence and constipation than the general population. There is often a fine dividing line between the two conditions, with any management intended to ameliorate, one risking precipitating the other. Bowel problems are observed to be the cause of much anxiety and may reduce quality of life in these people. Current bowel management is largely empirical with a limited research base. OBJECTIVES: To determine the effects of management strategies for faecal incontinence and constipation in people with neurological diseases affecting the central nervous system. SEARCH STRATEGY: We searched the Cochrane Incontinence Group Trials Register, the Cochrane Controlled Trials Register, MEDLINE, EMBASE and all reference lists of relevant articles. Date of the most recent searches: May 2000. SELECTION CRITERIA: All randomised or quasi-randomised trials evaluating any types of conservative, or surgical measure for the management of faecal incontinence and constipation in people with neurological diseases were selected. Specific therapies for the treatment of neurological diseases that indirectly affect bowel dysfunction have also been considered. DATA COLLECTION AND ANALYSIS: All three reviewers assessed the methodological quality of eligible trials and two reviewers independently extracted data from included trials using a range of pre-specified outcome measures. MAIN RESULTS: Only seven trials were identified by the search strategy and all were small and of poor quality. Oral medications for constipation were the subject of four trials. Cisapride does not seem to have clinically useful effects in people with spinal cord injuries (two trials). Psyllium was associated with increased stool frequency in people with Parkinson's disease but not altered colonic transit time (one trial). Some rectal preparations to initiate defecation produced faster results than others (one trial). Different time schedules for administration of rectal medication may produce different bowel responses (one trial). Mechanical evacuation may be more effective than oral or rectal medication (one trial). The clinical significance of any of these results is difficult to interpret. REVIEWER'S CONCLUSIONS: It is not possible to draw any recommendation for bowel care in people with neurological diseases from the trials included in this review. Bowel management for these people must remain empirical until well-designed controlled trials with adequate numbers and clinically relevant outcome measures become available.