Humberto Mestre

Immunization with neural-derived antigens inhibits lipid peroxidation after spinal cord injury

Lipid peroxidation (LP) is one of the most harmful mechanisms developed after spinal cord (SC) injury. Several strategies have been explored in order to control this phenomenon. Protective autoimmunity is a physiological process based on the modulation of inflammatory cells that can be boosted by immunizing with neural-derived peptides, such as A91. Since inflammatory cells are among the main contributors to lipid peroxidation, we hypothesized that protective autoimmunity could reduce LP after SC injury. In order to test this hypothesis, we designed two experiments in SC contused rats. First, animals were immunized with a neural-derived peptide seven days before injury. With the aim of inducing the functional elimination of CNS-specific T cells, for the second experiment, animals were tolerized against SC-protein extract and thereafter subjected to a SC injury. The lipid-soluble fluorescent products were used as an index of lipid peroxidation and were assessed after injury. Immunization with neural-derived peptides reduced lipid peroxidation after SC injury. Functional elimination of CNS-specific T cells avoided the beneficial effect induced by protective autoimmunity. The present study demonstrates the beneficial effect of immunizing with neural-derived peptides on lipid peroxidation inhibition; besides this, it also provides evidence on the neuroprotective mechanisms exerted by protective autoimmunity.

Immunization with A91 peptide or copolymer-1 reduces the production of nitric oxide and inducible nitric oxide synthase gene expression after spinal cord injury

Immunization with neurally derived peptides (INDP) boosts the action of an autoreactive immune response that has been shown to induce neuroprotection in several neurodegenerative diseases, especially after spinal cord (SC) injury. This strategy provides an environment that promotes neuronal survival and tissue preservation. The mechanisms by which this autoreactive response exerts its protective effects is not totally understood at the moment. A recent study showed that INDP reduces lipid peroxidation. Lipid peroxidation is a neurodegenerative phenomenon caused by the increased production of reactive nitrogen species such as nitric oxide (NO). It is possible that INDP could be interfering with NO production. To test this hypothesis, we examined the effect of INDP on the amount of NO produced by glial cells when cocultured with autoreactive T cells. We also evaluated the amount of NO and the expression of the inducible form of nitric oxide synthase (iNOS) at the injury site of SC‐injured animals. The neural‐derived peptides A91 and Cop‐1 were used to immunize mice and rats with SC injury. In vitro studies showed that INDP significantly reduces the production of NO by glial cells. This observation was substantiated by in vivo experiments demonstrating that INDP decreases the amount of NO and iNOS gene expression at the site of injury. The present study provides substantial evidence on the inhibitory effect of INDP on NO production, helpingour understanding of the mechanisms through which protective autoimmunity promotes neuroprotection.

Development of Protective Autoimmunity by Immunization with a Neural-Derived Peptide Is Ineffective in Severe Spinal Cord Injury

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 (TA91) 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 TA91 cells which predominantly showed to have a Th2 phenotype capable of producing BDNF, further promoting neuroprotection.

Immunization with a Neural-Derived Peptide Protects the Spinal Cord from Apoptosis after Traumatic Injury

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.

Lewis, Fischer 344, and sprague-dawley rats display differences in lipid peroxidation, motor recovery, and rubrospinal tract preservation after spinal cord injury.

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.

Copolymer-1 promotes neurogenesis and improves functional recovery after acute ischemic stroke in rats.

Stroke triggers a systemic inflammatory response that exacerbates the initial injury. Immunizing with peptides derived from CNS proteins can stimulate protective autoimmunity (PA). The most renowned of these peptides is copolymer-1 (Cop-1) also known as glatiramer acetate. This peptide has been approved for use in the treatment of multiple sclerosis. Cop-1-specific T cells cross the blood-brain barrier and secrete neurotrophins and anti-inflammatory cytokines that could stimulate proliferation of neural precursor cells and recruit them to the injury site; making it an ideal therapy for acute ischemic stroke. The aim of this work was to evaluate the effect of Cop-1 on neurogenesis and neurological recovery during the acute phase (7 days) and the chronic phase of stroke (60 days) in a rat model of transient middle cerebral artery occlusion (tMCAo). BDNF and NT-3 were quantified and infarct volumes were measured. We demonstrated that Cop-1 improves neurological deficit, enhances neurogenesis (at 7 and 60 days) in the SVZ, SGZ, and cerebral cortex through an increase in NT-3 production. It also decreased infarct volume even at the chronic phase of tMCAo. The present manuscript fortifies the support for the use of Cop-1 in acute ischemic stroke.

Prophylactic neuroprotection with A91 improves the outcome of spinal cord injured rats

Iatrogenic injury to the spinal cord (SC) is not an uncommon complication of spinal surgery. In an attempt to establish a preventive therapy for anticipated SC injury, we tested the effect of a single dose (SD) vaccine vs. the addition of a booster dose (BD) of a neural-derived peptide (A91) prior to SC contusion. Immunization with A91 immediately after SC injury has demonstrated to induce significant tissue protection and motor recovery. After injury, only the BD vaccination schedule had a neuroprotective effect. It was capable of improving neurological recovery that was always significantly higher than the one observed in rats with SD immunization or those only treated with PBS. Toward the end of study, animals treated with an A91 BD presented a BBB score of 9.75±0.17 (mean±standard deviation) while rats treated with SD or PBS had a score of 6.6±0.7 and 5.6±0.6 respectively. In the next step we attempted to corroborate the neuroprotective effect induced by A91 immunization. For this purpose, we assessed the survival of rubrospinal neurons (RSNs) and ventral horn neurons (VHNs) sixty days after SC injury. BD vaccination induced a significant survival of both RSNs and VHNs after injury. Finally, the failure or success of this therapy (SD or BD respectively) was associated with a lower (SD) or higher (BD) A91-specific T cell proliferation. Prophylactic neuroprotection with an initial and subsequent booster dose of A91 may improve recovery after SC injury sustained during invasive spinal surgery procedures.

Therapeutic Window for Combination Therapy of A91 Peptide and Glutathione Allows Delayed Treatment After Spinal Cord Injury

Immunisation with neural‐derived peptides is a promising strategy in models of spinal cord (SC) injury. Recent studies have also demonstrated that the addition of glutathione monoethyl ester (GHSE) to this strategy further improves motor recovery, tissue protection and neuronal survival after SC injury. As it is realistic to envision that this combination therapy could be tested in clinical trials, the therapeutic window should be experimentally explored before implementing its use in SC‐injured human beings. For this purpose, 50 rats (10 per group) were subjected to a moderate SC contusion. The combined therapy was initiated at 10 min., 24, 72 or 120 hr after injury. Motor recovery and the survival of rubrospinal (RS) and ventral horn (VH) neurones were evaluated 60 days after injury. Results showed a significant motor improvement even if the combined therapy was initiated up to 72 hr after injury. BBB scores were as follows: 10 min.: 10.5 ± 0.7, 24 hr: 10.7 ± 0.5, 72 hr: 11.0 ± 1.3 and PBS: 6.7 ± 1 (mean ± S.D.). Initiation of combined therapy 120 hr after injury had no beneficial effect on motor recovery. Survival of RS and VH neurones was significantly higher in animals treated during the first 72 hr than those treated only with PBS. In this case again, animals treated with combined therapy 120 hr after injury did not present significant survival of neurones. Treatment with this combined strategy has a clinically feasible therapeutic window. This therapy provides enough time to transport and diagnose the patient and allows the concomitant use of other neuroprotective therapies.

Immunization with Neural-Derived Peptides as a Potential Therapy in Neurodegenerative Diseases

There is a nosological dilemma when it comes to classifying what comprises a neurodegenerative disease (NDD). Degeneration – purely speaking – is to go from a higher to a lower level of functioning; it is deterioration from normalcy. Neurons are the functional elements of the nervous system. Then degeneration of the nervous system consists of a decrease or loss in the function of neurons. Not necessarily an atrophy, which consists of the death of a particular population of neurons. Clinically, NDD are comprised of progressive dementias, progressive ataxias, disorders in posture and movement, muscle weakness, and progressive blindness. The common characteristic in all of these pathologies is their chronicity. Each and every one of the aforementioned diseases consists of a chronic progression towards the loss of a particular function. However, this definition does not include a limit on temporality. Nosologically speaking neurodegeneration could include several other pathologies from an acute time frame. NDD can further be divided into an acute and chronic classification. Chronic diseases such as: amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD) and Parkinson disease (PD) were the common conception of NDD. The latter was sustained until acute traumatic injuries to the central nervous system (CNS) were found to cause generalized inflammation and other phenomena that lead to degeneration. Examples of CNS injury that cause this secondary degeneration are: global or focal cerebral ischemia (stroke), spinal cord injury (SCI), and traumatic brain injury (TBI). The similarities in neurodegenerative processes between these and chronic NDD allows us to classify them within acute NDD. Neurodegeneration previously consisted of progressive atrophic disorders but has now expanded into the study of all pathophysiological processes that deteriorate the CNS. As a whole, NDD are the cause of many deaths around the world. In the US, stroke, traumatic injuries (such as: SCI and TBI), AD, and PD are within the top 15 causes of mortality, averaging 350,000 deaths per year (Xu et al., 2007). Although NDD have an elevated mortality their greatest impact is on morbidity, affecting 50 million Americans each year and generating a large amount of federal spending (Brown et al., 2005). Every year

Pharmacological Treatment of Acute Ischemic Stroke

144 billion USD are spent on AD alone, and that is excluding the spending required for the other 600 neurological disorders that have been described (Alzheimer’s Association, 2010; Meek et al., 1998). The elevated prevalence and incidence require a large initiative to research the hallmarks of these diseases. Until now, our understanding of NDD is quite