Roberta Barbizan

Motor Recovery and Synaptic Preservation after Ventral Root Avulsion and Repair with a Fibrin Sealant Derived from Snake Venom

Background Ventral root avulsion is an experimental model of proximal axonal injury at the central/peripheral nervous system interface that results in paralysis and poor clinical outcome after restorative surgery. Root reimplantation may decrease neuronal degeneration in such cases. We describe the use of a snake venom-derived fibrin sealant during surgical reconnection of avulsed roots at the spinal cord surface. The present work investigates the effects of this fibrin sealant on functional recovery, neuronal survival, synaptic plasticity, and glial reaction in the spinal motoneuron microenvironment after ventral root reimplantation. Methodology/Principal Findings Female Lewis rats (7 weeks old) were subjected to VRA and root replantation. The animals were divided into two groups: 1) avulsion only and 2) replanted roots with fibrin sealant derived from snake venom. Post-surgical motor performance was evaluated using the CatWalk system twice a week for 12 weeks. The rats were sacrificed 12 weeks after surgery, and their lumbar intumescences were processed for motoneuron counting and immunohistochemistry (GFAP, Iba-1 and synaptophysin antisera). Array based qRT-PCR was used to evaluate gene regulation of several neurotrophic factors and receptors as well as inflammatory related molecules. The results indicated that the root reimplantation with fibrin sealant enhanced motor recovery, preserved the synaptic covering of the motoneurons and improved neuronal survival. The replanted group did not show significant changes in microglial response compared to VRA-only. However, the astroglial reaction was significantly reduced in this group. Conclusions/Significance In conclusion, the present data suggest that the repair of avulsed roots with snake venom fibrin glue at the exact point of detachment results in neuroprotection and preservation of the synaptic network at the microenvironment of the lesioned motoneurons. Also such procedure reduced the astroglial reaction and increased mRNA levels to neurotrophins and anti-inflammatory cytokines that may in turn, contribute to improving recovery of motor function.

Impact of acute inflammation on spinal motoneuron synaptic plasticity following ventral root avulsion.

BackgroundVentral root avulsion is a proximal nerve root lesion in which ventral motor nerve rootlets are torn from surface of the spinal cord, resulting in extensive death of motoneurons. It has been previously shown that if such lesioning is performed in an animal with experimental autoimmune encephalomyelitis (EAE), a significant number of motoneurons can be rescued despite an intense inflammatory reaction. This rescue effect has been attributed to production of a number of neurotrophic factors by invading T cells. Synaptological changes may be involved in neuronal degeneration, and a better understanding of the role of these changes may be of importance for developing new strategies to promote neuronal survival. The objective of the present work was to evaluate neuronal survival, astroglial reaction and synaptic input changes in spinal cord anterior horn motor nuclei after ventral root avulsion in animals with EAE, both during peak disease and after remission.MethodsLewis rats were subjected to unilateral avulsion of lumbar ventral roots (VRA) and divided into three groups: VRA control, VRA at peak of EAE, and VRA during EAE remission. The animals were sacrificed and their lumbar spinal cords processed for immunohistochemistry, transmission electron microscopy, and motoneuron counting.ResultsThe results indicate a reduction in astroglial reaction, a maintenance of microglial reactivity, and increases in synaptic covering of, and survival of, motoneurons in the VRA+EAE group as compared to VRA alone.ConclusionThe present findings indicate that CNS inflammation may directly influence synaptic plasticity as well as the stability of neuronal networks, positively influencing the survival of lesioned neurons.

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.

Synaptic plasticity and sensory-motor improvement following fibrin sealant dorsal root reimplantation and mononuclear cell therapy

Root lesions may affect both dorsal and ventral roots. However, due to the possibility of generating further inflammation and neuropathic pain, surgical procedures do not prioritize the repair of the afferent component. The loss of such sensorial input directly disturbs the spinal circuits thus affecting the functionality of the injuried limb. The present study evaluated the motor and sensory improvement following dorsal root reimplantation with fibrin sealant (FS) plus bone marrow mononuclear cells (MC) after dorsal rhizotomy. MC were used to enhance the repair process. We also analyzed changes in the glial response and synaptic circuits within the spinal cord. Female Lewis rats (6–8 weeks old) were divided in three groups: rhizotomy (RZ group), rhizotomy repaired with FS (RZ+FS group) and rhizotomy repaired with FS and MC (RZ+FS+MC group). The behavioral tests electronic von-Frey and Walking track test were carried out. For immunohistochemistry we used markers to detect different synapse profiles as well as glial reaction. The behavioral results showed a significant decrease in sensory and motor function after lesion. The reimplantation decreased glial reaction and improved synaptic plasticity of afferent inputs. Cell therapy further enhanced the rewiring process. In addition, both reimplanted groups presented twice as much motor control compared to the non-treated group. In conclusion, the reimplantation with FS and MC is efficient and may be considered an approach to improve sensory-motor recovery following dorsal rhizotomy.

Granulocyte colony stimulating factor neuroprotective effects on spinal motoneurons after ventral root avulsion

G‐CSF is a glycoprotein commonly used to treat neutropenia. Recent studies have shown that the G‐CSF receptor (G‐CSF‐R) is expressed by neurons in the central nervous system (CNS), and neuroprotective effects of G‐CSF have been observed. In this study, the influence of G‐CSF treatment on the glial reactivity and synaptic plasticity of spinal motoneurons in rats subjected to ventral root avulsion (VRA) was investigated. Lewis rats (7 weeks old) were subjected to unilateral VRA and divided into two groups: G‐CSF and placebo treated. The drug treated animals were injected subcutaneously with 200 μg/kg/day of G‐CSF for 5 days post lesion. The placebo group received saline buffer. After 2 weeks, both groups were sacrificed and their lumbar intumescences processed for transmission electron microscopy (TEM), motoneuron counting, and immunohistochemistry with antibodies against GFAP, Iba‐1, and synaptophysin. Furthermore, in vitro analysis was carried out, using newborn cortical derived astrocytes. The results indicated increased neuronal survival in the G‐CSF treated group coupled with synaptic preservation. TEM analyses revealed an improved preservation of the synaptic covering in treated animals. Additionally, the drug treated group showed an increase in astroglial reactivity both in vivo and in vitro. The astrocytes also presented an increased cell proliferation rate when compared with the controls after 3 days of culturing. In conclusion, the present results suggest that G‐CSF has an influence on the stability of presynaptic terminals in the spinal cord as well as on the astroglial reaction, indicating a possible neuroprotective action. Synapse, 2012.

Multiple uses of fibrin sealant for nervous system treatment following injury and disease

Lesions to the nervous system often produce hemorrhage and tissue loss that are difficult, if not impossible, to repair. Therefore, scar formation, inflammation and cavitation take place, expanding the lesion epicenter. This significantly worsens the patient conditions and impairment, increasing neuronal loss and glial reaction, which in turn further decreases the chances of a positive outcome. The possibility of using hemostatic substances that also function as a scaffold, such as the fibrin sealant, reduces surgical time and improve postoperative recovery. To date, several studies have demonstrated that human blood derived fibrin sealant produces positive effects in different interventions, becoming an efficient alternative to suturing. To provide an alternative to homologous fibrin sealants, the Center for the Study of Venoms and Venomous Animals (CEVAP, Brazil) has proposed a new bioproduct composed of certified animal components, including a thrombin-like enzyme obtained from snake venom and bubaline fibrinogen. Thus, the present review brings up to date literature assessment on the use of fibrin sealant for nervous system repair and positions the new heterologous bioproduct from CEVAP as an alternative to the commercial counterparts. In this way, clinical and pre-clinical data are discussed in different topics, ranging from central nervous system to peripheral nervous system applications, specifying positive results as well as future enhancements that are necessary for improving the use of fibrin sealant therapy.

Long-Term Spinal Ventral Root Reimplantation, but not Bone Marrow Mononuclear Cell Treatment, Positively Influences Ultrastructural Synapse Recovery and Motor Axonal Regrowth

We recently proposed a new surgical approach to treat ventral root avulsion, resulting in motoneuron protection. The present work combined such a surgical approach with bone marrow mononuclear cells (MC) therapy. Therefore, MC were added to the site of reimplantation. Female Lewis rats (seven weeks old) were subjected to unilateral ventral root avulsion (VRA) at L4, L5 and L6 levels and divided into the following groups (n = 5 for each group): Avulsion, sealant reimplanted roots and sealant reimplanted roots plus MC. After four weeks and 12 weeks post-surgery, the lumbar intumescences were processed by transmission electron microscopy, to analyze synaptic inputs to the repaired α motoneurons. Also, the ipsi and contralateral sciatic nerves were processed for axon counting and morphometry. The ultrastructural results indicated a significant preservation of inhibitory pre-synaptic boutons in the groups repaired with sealant alone and associated with MC therapy. Moreover, the average number of axons was higher in treated groups when compared to avulsion only. Complementary to the fiber counting, the morphometric analysis of axonal diameter and “g” ratio demonstrated that root reimplantation improved the motor component recovery. In conclusion, the data herein demonstrate that root reimplantation at the lesion site may be considered a therapeutic approach, following proximal lesions in the interface of central nervous system (CNS) and peripheral nervous system (PNS), and that MC therapy does not further improve the regenerative recovery, up to 12 weeks post lesion.

Expressão do complexo de histocompatilidade principal de classe I (MHC I) no sistema nervoso central: plasticidade sináptica e regeneração

It has been recently demonstrated that the major histocompatibility complex of class I (MHC I) expressed in the central nervous system (CNS) does not only function as a molecule of the immune system, but also plays a role in the synaptic plasticity. The expression of MHC I influences the intensity and selectivity of elimination of synapses apposed to neurons that were subjected to lesion, besides influencing the reactivity of neighboring glial cells. MHC I expression and the degree of synaptic rearrangement and glial response after injury correlate with differences in the regenerative potential and functional recovery of isogenic mice strains. In this way, the new aspects regarding MHC I functions in the CNS may guide further studies aiming at searching the involvement of MCH I in neurologic disorders, as well as the development of new therapeutic strategies.Foi demonstrado recentemente que o complexo de histocompatibilidade principal de classe I (MHC I), expresso no sistema nervoso central (SNC), nao funciona somente como molecula com papel imunologico, mas tambem como parte de um mecanismo envolvido na plasticidade sinaptica. A expressao de MHC I interfere na intensidade e seletividade da retracao de sinapses em contato com neuronios que sofreram lesao e tambem influencia a reatividade das celulas gliais proximas a esses neuronios. A intensidade do rearranjo sinaptico e resposta glial apos lesao, ligadas a expressao de MHC I no SNC, repercute em diferencas na capacidade regenerativa e recuperacao funcional em linhagens de camundongos isogenicos. Dessa forma, os novos aspectos sobre a funcao do MHC I no SNC direcionam futuras pesquisas no sentido de buscar o envolvimento do MHC I em doencas neurologicas e tambem o desenvolvimento de novas estrategias terapeuticas.

Direct Spinal Ventral Root Repair following Avulsion: Effectiveness of a New Heterologous Fibrin Sealant on Motoneuron Survival and Regeneration.

Axonal injuries at the interface between central and peripheral nervous system, such as ventral root avulsion (VRA), induce important degenerative processes, mostly resulting in neuronal and motor function loss. In the present work, we have compared two different fibrin sealants, one derived from human blood and another derived from animal blood and Crotalus durissus terrificus venom, as a promising treatment for this type of injury. Lewis rats were submitted to VRA (L4–L6) and had the avulsed roots reimplanted to the surface of the spinal cord, with the aid of fibrin sealant. The spinal cords were processed to evaluate neuronal survival, synaptic stability, and glial reactivity, 4 and 12 weeks after lesion. Sciatic nerves were processed to investigate Schwann cell activity by p75NTR expression (4 weeks after surgery) and to count myelinated axons and morphometric evaluation (12 weeks after surgery). Walking track test was used to evaluate gait recovery, up to 12 weeks. The results indicate that both fibrin sealants are similarly efficient. However, the snake-derived fibrin glue is a potentially safer alternative for being a biological and biodegradable product which does not contain human blood derivatives. Therefore, the venom glue can be a useful tool for the scientific community due to its advantages and variety of applications.

Expression of class I major histocompatibility complex (MHC I) in the central nervous system: role in synaptic plasticity and regeneration

It has been recently demonstrated that the major histocompatibility complex of class I (MHC I) expressed in the central nervous system (CNS) does not only function as a molecule of the immune system, but also plays a role in the synaptic plasticity. The expression of MHC I influences the intensity and selectivity of elimination of synapses apposed to neurons that were subjected to lesion, besides influencing the reactivity of neighboring glial cells. MHC I expression and the degree of synaptic rearrangement and glial response after injury correlate with differences in the regenerative potential and functional recovery of isogenic mice strains. In this way, the new aspects regarding MHC I functions in the CNS may guide further studies aiming at searching the involvement of MCH I in neurologic disorders, as well as the development of new therapeutic strategies.Foi demonstrado recentemente que o complexo de histocompatibilidade principal de classe I (MHC I), expresso no sistema nervoso central (SNC), nao funciona somente como molecula com papel imunologico, mas tambem como parte de um mecanismo envolvido na plasticidade sinaptica. A expressao de MHC I interfere na intensidade e seletividade da retracao de sinapses em contato com neuronios que sofreram lesao e tambem influencia a reatividade das celulas gliais proximas a esses neuronios. A intensidade do rearranjo sinaptico e resposta glial apos lesao, ligadas a expressao de MHC I no SNC, repercute em diferencas na capacidade regenerativa e recuperacao funcional em linhagens de camundongos isogenicos. Dessa forma, os novos aspectos sobre a funcao do MHC I no SNC direcionam futuras pesquisas no sentido de buscar o envolvimento do MHC I em doencas neurologicas e tambem o desenvolvimento de novas estrategias terapeuticas.