Combination of a Gellan Gum-Based Hydrogel With Cell Therapy for the Treatment of Cervical Spinal Cord Injury.

Cervical spinal cord trauma represents more than half of the spinal cord injury (SCI) cases worldwide. Respiratory compromise, as well as severe limb motor deficits, are among the main consequences of cervical lesions. In the present work, a Gellan Gum (GG)-based hydrogel modified with GRGDS peptide, together with adipose tissue-derived stem/stromal cells (ASCs) and olfactory ensheathing cells (OECs), was used as a therapeutic strategy after a C2 hemisection SCI in rats. Hydrogel or cells alone, and a group without treatment, were also tested. Four weeks after injury, compound muscle action potentials (CMAPs) were performed to assess functional phrenic motor neuron (PhMN) innervation of the diaphragm; no differences were observed amongst groups, confirming that the PhMN pool located between C3 and C5 was not affected by the C2 injury or by the treatments. In the same line, the vast majority of diaphragmatic neuromuscular junctions remained intact. Five weeks post-injury, inspiratory bursting of the affected ipsilateral hemidiaphragm was evaluated through EMG recordings of dorsal, medial and ventral subregions of the muscle. All treatments significantly increased EMG amplitude at the ventral portion in comparison to untreated animals, but only the combinatorial group presented increased EMG amplitude at the medial portion of the hemidiaphragm. No differences were observed in forelimb motor function, neither in markers for axonal regrowth (neuronal tracers), astrogliosis (GFAP) and inflammatory cells (CD68). Moreover, using Von Frey testing of mechanical allodynia, it was possible to find a significant effect of the group combining hydrogel and cells on hypersensitivity; rats with a SCI displayed an increased response of the contralateral forelimb to a normally innocuous mechanical stimulus, but after treatment with the combinatorial therapy this behavior was reverted almost to the levels of uninjured controls. These results suggest that our therapeutic approach may have beneficial effects on both diaphragmatic recovery and sensory function.

Long-Distance Axon Regeneration Promotes Recovery of Diaphragmatic Respiratory Function after Spinal Cord Injury.

Compromise in inspiratory breathing following cervical spinal cord injury (SCI) is caused by damage to descending bulbospinal axons originating in the rostral ventral respiratory group (rVRG) and consequent denervation and silencing of phrenic motor neurons (PhMNs) that directly control diaphragm activation. In a rat model of high-cervical hemisection SCI, we performed systemic administration of an antagonist peptide directed against phosphatase and tensin homolog (PTEN), a central inhibitor of neuron-intrinsic axon growth potential. PTEN antagonist peptide (PAP4) robustly restored diaphragm function, as determined with electromyography (EMG) recordings in living SCI animals. PAP4 promoted substantial, long-distance regeneration of injured rVRG axons through the lesion and back toward PhMNs located throughout the C3-C5 spinal cord. These regrowing rVRG axons also formed putative excitatory synaptic connections with PhMNs, demonstrating reconnection of rVRG-PhMN-diaphragm circuitry. Lastly, re-lesion through the hemisection site completely ablated functional recovery induced by PAP4. Collectively, our findings demonstrate that axon regeneration in response to systemic PAP4 administration promoted recovery of diaphragmatic respiratory function after cervical SCI.

Astrocyte progenitor transplantation promotes regeneration of bulbospinal respiratory axons, recovery of diaphragm function, and a reduced macrophage response following cervical spinal cord injury.

Stem/progenitor cell transplantation delivery of astrocytes is a potentially powerful strategy for spinal cord injury (SCI). Axon extension into SCI lesions that occur spontaneously or in response to experimental manipulations is often observed along endogenous astrocyte "bridges," suggesting that augmenting this response via astrocyte lineage transplantation can enhance axon regrowth. Given the importance of respiratory dysfunction post-SCI, we transplanted glial-restricted precursors (GRPs)-a class of lineage-restricted astrocyte progenitors-into the C2 hemisection model and evaluated effects on diaphragm function and the growth response of descending rostral ventral respiratory group (rVRG) axons that innervate phrenic motor neurons (PhMNs). GRPs survived long term and efficiently differentiated into astrocytes in injured spinal cord. GRPs promoted significant recovery of diaphragm electromyography amplitudes and stimulated robust regeneration of injured rVRG axons. Although rVRG fibers extended across the lesion, no regrowing axons re-entered caudal spinal cord to reinnervate PhMNs, suggesting that this regeneration response-although impressive-was not responsible for recovery. Within ipsilateral C3-5 ventral horn (PhMN location), GRPs induced substantial sprouting of spared fibers originating in contralateral rVRG and 5-HT axons that are important for regulating PhMN excitability; this sprouting was likely involved in functional effects of GRPs. Finally, GRPs reduced the macrophage response (which plays a key role in inducing axon retraction and limiting regrowth) both within the hemisection and at intact caudal spinal cord surrounding PhMNs. These findings demonstrate that astrocyte progenitor transplantation promotes significant plasticity of rVRG-PhMN circuitry and restoration of diaphragm function and suggest that these effects may be in part through immunomodulation.

Calcineurin Dysregulation Underlies Spinal Cord Injury-Induced K+ Channel Dysfunction in DRG Neurons.

Dysfunction of the fast-inactivating Kv3.4 potassium current in dorsal root ganglion (DRG) neurons contributes to the hyperexcitability associated with persistent pain induced by spinal cord injury (SCI). However, the underlying mechanism is not known. In light of our previous work demonstrating modulation of the Kv3.4 channel by phosphorylation, we investigated the role of the phosphatase calcineurin (CaN) using electrophysiological, molecular, and imaging approaches in adult female Sprague Dawley rats. Pharmacological inhibition of CaN in small-diameter DRG neurons slowed repolarization of the somatic action potential (AP) and attenuated the Kv3.4 current. Attenuated Kv3.4 currents also exhibited slowed inactivation. We observed similar effects on the recombinant Kv3.4 channel heterologously expressed in Chinese hamster ovary cells, supporting our findings in DRG neurons. Elucidating the molecular basis of these effects, mutation of four previously characterized serines within the Kv3.4 N-terminal inactivation domain eliminated the effects of CaN inhibition on the Kv3.4 current. SCI similarly induced concurrent Kv3.4 current attenuation and slowing of inactivation. Although there was little change in CaN expression and localization after injury, SCI induced upregulation of the native regulator of CaN 1 (RCAN1) in the DRG at the transcript and protein levels. Consistent with CaN inhibition resulting from RCAN1 upregulation, overexpression of RCAN1 in naive DRG neurons recapitulated the effects of pharmacological CaN inhibition on the Kv3.4 current and the AP. Overall, these results demonstrate a novel regulatory pathway that links CaN, RCAN1, and Kv3.4 in DRG neurons. Dysregulation of this pathway might underlie a peripheral mechanism of pain sensitization induced by SCI.SIGNIFICANCE STATEMENT Pain sensitization associated with spinal cord injury (SCI) involves poorly understood maladaptive modulation of neuronal excitability. Although central mechanisms have received significant attention, recent studies have identified peripheral nerve hyperexcitability as a driver of persistent pain signaling after SCI. However, the ion channels and signaling molecules responsible for this change in primary sensory neuron excitability are still not well defined. To address this problem, this study used complementary electrophysiological and molecular methods to determine how Kv3.4, a voltage-gated K+ channel robustly expressed in dorsal root ganglion neurons, becomes dysfunctional upon calcineurin (CaN) inhibition. The results strongly suggest that CaN inhibition underlies SCI-induced dysfunction of Kv3.4 and the associated excitability changes through upregulation of the native regulator of CaN 1 (RCAN1).

[Study on Portuguese Medical Schools' Learning Conditions: A National Analysis on Student Satisfaction, Student-Tutor Ratios and Number of Admissions]. / Estudo sobre as Condições Pedagógicas das Escolas Médicas Portuguesas: Uma Análise Nacional Sobre a Satisfação Estudantil, Rácios Estudante-Tutor e Número de Admissões.

INTRODUCTION: Experiences of clinical and nonclinical learning environments, as well as assessment and study environments influence student satisfaction with their medical schools. Student-tutor ratios may impact on their perception of clinical learning environments. The aim of this study was to analyze medical students' satisfaction and student-tutor ratios in relation to medical schools' number of admissions. MATERIALS AND METHODS: A questionnaire was created, regarding learning, assessment and study environments in eight medical schools. 2037 students participated in this cross-sectional study. Cronbach' alpha (internal consistency) was calculated and principal component analysis was conducted. Pearson correlations and multiple comparisons were analyzed. RESULTS: Assessment environments showed the highest satisfaction scores and clinical learning environments the lowest scores. The national student-tutor ratio in clinical rotations is 7.53; there are significant differences among schools. Institutions with higher number of admissions showed the lowest scores of overall student satisfaction (r = -0.756; p < 0.05), which decreased with progression in the medical course. High student-tutor ratios are strongly correlated with low levels of satisfaction regarding clinical learning environments (r = -0.826; p < 0.05). DISCUSSION: Clinical learning environments show the lowest satisfaction scores, which may expose the effect of high ratios in clinical rotations. Depending on the number of admissions, significant differences between medical schools were found. Quality of teaching-learning strategies and articulation with hospitals might also be important variables. CONCLUSION: Medical schools with more admissions might be more susceptible to lower scores of student satisfaction. High student-tutor ratios in clinical rotations may reduce the quality of learning experiences and inhibit the acquisition of competences.

Introdução: Os ambientes de ensino clínico e não clínico, bem como as condições de avaliação e estudo, influenciam a satisfação estudantil com as Escolas Médicas. Os rácios estudante-tutor podem ter impacto na perceção sobre o ensino em meio clínico. Este estudo tem como objetivo analisar a satisfação dos estudantes de Medicina e os rácios estudante-tutor em relação com o número de admissões das Escolas Médicas. Materiais e Métodos: Foi criado um questionário sobre os ambientes de aprendizagem, avaliação e estudo em oito Escolas Médicas, distribuído a 2037 estudantes. Calculou-se o alfa de Cronbach (consistência interna) e executou-se uma análise de componentes principais. Resultados: Condições de avaliação obtiveram os melhores resultados de satisfação, enquanto o ensino em meio clínico revelou as menores pontuações. O rácio estudante-tutor nacional em disciplinas clínicas (7,53) traduz diferenças significativas entre Escolas. Instituições com maior número de admissões evidenciam resultados inferiores de satisfação estudantil (r= -0,756; p < 0,05), com redução progressiva ao longo do curso. Elevados rácios estão correlacionados com baixa satisfação com o ensino em meio clínico (r= -0,826; p < 0,05). Discussão: O ensino em meio clínico evidencia menor satisfação estudantil, traduzindo os elevados rácios em disciplinas clínicas. Dependendo do número de admissões, existem diferenças significativas entre Escolas. A qualidade das estratégias de ensino-aprendizagem e articulação hospitalar podem igualmente ser variáveis importantes. Conclusão: As Escolas com maior número de admissões podem ser mais suscetíveis a baixos resultados de satisfação estudantil. Elevados rácios estudante-tutor em disciplinas clínicas podem reduzir a qualidade do ensino em meio clínico e inibir a aquisição de competências.

iPS cell transplantation for traumatic spinal cord injury.

A large body of work has been published on transplantation of a wide range of neural stem and progenitor cell types derived from the developing and adult CNS, as well as from pluripotent embryonic stem cells, in models of traumatic spinal cord injury (SCI). However, many of these cell-based approaches present practical issues for clinical translation such as ethical cell derivation, generation of potentially large numbers of homogenously prepared cells, and immune rejection. With the advent of induced Pluripotent Stem (iPS) cell technology, many of these issues may potentially be overcome. To date, a number of studies have demonstrated integration, differentiation into mature CNS lineages, migration and long-term safety of iPS cell transplants in a variety of SCI models, as well as therapeutic benefits in some cases. Given the clinical potential of this advance in stem cell biology, we present a concise review of studies published to date involving iPS cell transplantation in animal models of SCI.