RESUMO
The rat is the most common animal model for the preclinical validation of neuroprotective therapies in spinal cord injury (SCI). Lipid peroxidation (LP) is a hallmark of the damage triggered after SCI. Free radicals react with fatty acids causing cellular and membrane disruption. LP accounts for a considerable amount of neuronal cell death after SCI. To better understand the implications of inbred and outbred rat strain selection on preclinical SCI research, we evaluated LP after laminectomy sham surgery and a severe contusion of the T9 spinal cord in female Sprague-Dawley (SPD), Lewis (LEW), and Fischer 344 (F344) rats. Further analysis included locomotor recovery using the Basso, Beattie, and Bresnahan (BBB) scale and retrograde rubrospinal tract tracing. LEW had the highest levels of LP products 72 h after sham surgery and SCI, significantly different from both F344 and SPD. SPD rats had the fastest functional recovery and highest BBB scores; these were not significantly different to F344. However, LEW rats achieved the lowest BBB scores throughout the 2-month follow-up, yielding significant differences when compared to SPD and F344. To see if the improvement in locomotion was secondary to an increase in axon survival, we evaluated rubrospinal neurons (RSNs) via retrograde labeling of the rubrospinal tract and quantified cells at the red nuclei. The highest numbers of RSNs were observed in SPD rats then F344; the lowest counts were seen in LEW rats. The BBB scores significantly correlated with the amount of positively stained RSN in the red nuclei. It is critical to identify interstrain variations as a potential confound in preclinical research. Multi-strain validation of neuroprotective therapies may increase chances of successful translation.
RESUMO
Monocyte locomotion inhibitory factor (MLIF) is a pentapeptide produced by Entamoeba histolytica that has a potent anti-inflammatory effect. Either MLIF or phosphate buffered saline (PBS) was administered directly onto the spinal cord (SC) immediately after injury. Motor recovery was evaluated. We also analyzed neuroprotection by quantifying the number of surviving ventral horn motor neurons and the persistence of rubrospinal tract neurons. To evaluate the mechanism through which MLIF improved the outcome of SC injury, we quantified the expression of inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10), and transforming growth factor- ß (TGF- ß ) genes at the site of injury. Finally, the levels of nitric oxide and of lipid peroxidation were also determined in peripheral blood. Results showed that MLIF improved the rate of motor recovery and this correlated with an increased survival of ventral horn and rubrospinal neurons. These beneficial effects were in turn associated with a reduction in iNOS gene products and a significant upregulation of IL-10 and TGF- ß expression. In the same way, MLIF reduced the concentration of nitric oxide and the levels of lipid peroxidation in systemic circulation. The present results demonstrate for the first time the neuroprotective effects endowed by MLIF after SC injury.
Assuntos
Entamoeba histolytica/química , Locomoção/efeitos dos fármacos , Oligopeptídeos/administração & dosagem , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Células do Corno Anterior/efeitos dos fármacos , Células do Corno Anterior/fisiopatologia , Humanos , Interleucina-10/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Óxido Nítrico Sintase Tipo II/metabolismo , Oligopeptídeos/química , Ratos , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Apoptosis is one of the most destructive mechanisms that develop after spinal cord (SC) injury. Immunization with neural-derived peptides (INDPs) such as A91 has shown to reduce the deleterious proinflammatory response and the amount of harmful compounds produced after SC injury. With the notion that the aforementioned elements are apoptotic inducers, we hypothesized that INDPs would reduce apoptosis after SC injury. In order to test this assumption, adult rats were subjected to SC contusion and immunized either with A91 or phosphate buffered saline (PBS; control group). Seven days after injury, animals were euthanized to evaluate the number of apoptotic cells at the injury site. Apoptosis was evaluated using DAPI and TUNEL techniques; caspase-3 activity was also evaluated. To further elucidate the mechanisms through which A91 exerts this antiapoptotic effects we quantified tumor necrosis factor-alpha (TNF-α). To also demonstrate that the decrease in apoptotic cells correlated with a functional improvement, locomotor recovery was evaluated. Immunization with A91 significantly reduced the number of apoptotic cells and decreased caspase-3 activity and TNF-α concentration. Immunization with A91 also improved the functional recovery of injured rats. The present study shows the beneficial effect of INDPs on preventing apoptosis and provides more evidence on the neuroprotective mechanisms exerted by this strategy.
Assuntos
Apoptose/efeitos dos fármacos , Imunização , Proteínas do Tecido Nervoso/farmacologia , Peptídeos/farmacologia , Traumatismos da Medula Espinal/imunologia , Animais , Apoptose/imunologia , Feminino , Proteínas do Tecido Nervoso/imunologia , Peptídeos/imunologia , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/patologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Protective autoimmunity (PA) is a physiological response to central nervous system trauma that has demonstrated to promote neuroprotection after spinal cord injury (SCI). To reach its beneficial effect, PA should be boosted by immunizing with neural constituents or neural-derived peptides such as A91. Immunizing with A91 has shown to promote neuroprotection after SCI and its use has proven to be feasible in a clinical setting. The broad applications of neural-derived peptides make it important to determine the main features of this anti-A91 response. For this purpose, adult Sprague-Dawley rats were subjected to a spinal cord contusion (SCC; moderate or severe) or a spinal cord transection (SCT; complete or incomplete). Immediately after injury, animals were immunized with PBS or A91. Motor recovery, T cell-specific response against A91 and the levels of IL-4, IFN-γ and brain-derived neurotrophic factor (BDNF) released by A91-specific T (T(A91)) cells were evaluated. Rats with moderate SCC, presented a better motor recovery after A91 immunization. Animals with moderate SCC or incomplete SCT showed significant T cell proliferation against A91 that was characterized chiefly by the predominant production of IL-4 and the release of BDNF. In contrast, immunization with A91 did not promote a better motor recovery in animals with severe SCC or complete SCT. In fact, T cell proliferation against A91 was diminished in these animals. The present results suggest that the effective development of PA and, consequently, the beneficial effects of immunizing with A91 significantly depend on the severity of SCI. This could mainly be attributed to the lack of T(A91) cells which predominantly showed to have a Th2 phenotype capable of producing BDNF, further promoting neuroprotection.