Catalytic antioxidant nanoparticles mitigate secondary injury progression and promote functional recovery in spinal cord injury model.

Traumatic spinal cord injury exacerbates disability with time due to secondary injury cascade triggered largely by overproduction of reactive oxygen species (ROS) at the lesion site, causing oxidative stress. This study explored nanoparticles containing antioxidant enzymes (antioxidant NPs) to neutralize excess ROS at the lesion site and its impact. When tested in a rat contusion model of spinal cord injury, a single dose of antioxidant NPs, administered intravenously three hours after injury, effectively restored the redox balance at the lesion site, interrupting the secondary injury progression. This led to reduced spinal cord tissue inflammation, apoptosis, cavitation, and inhibition of syringomyelia. Moreover, the treatment reduced scar tissue forming collagen at the lesion site, protected axons from demyelination, and stimulated lesion healing, with further analysis indicating the formation of immature neurons. The ultimate effect of the treatment was improved motor and sensory functions and rapid post-injury weight loss recovery. Histological analysis revealed activated microglia in the spinal cord displaying rod-shaped anti-inflammatory and regenerative phenotype in treated animals, contrasting with amoeboid inflammatory and degenerative phenotype in untreated control. Overall data suggest that restoring redox balance at the lesion site shifts the dynamics in the injured spinal cord microenvironment from degenerative to regenerative, potentially by promoting endogenous repair mechanisms. Antioxidant NPs show promise to be developed as an early therapeutic intervention in stabilizing injured spinal cord for enhanced recovery.

Antioxidant Therapy in Oxidative Stress-Induced Neurodegenerative Diseases: Role of Nanoparticle-Based Drug Delivery Systems in Clinical Translation.

Free radicals are formed as a part of normal metabolic activities but are neutralized by the endogenous antioxidants present in cells/tissue, thus maintaining the redox balance. This redox balance is disrupted in certain neuropathophysiological conditions, causing oxidative stress, which is implicated in several progressive neurodegenerative diseases. Following neuronal injury, secondary injury progression is also caused by excessive production of free radicals. Highly reactive free radicals, mainly the reactive oxygen species (ROS) and reactive nitrogen species (RNS), damage the cell membrane, proteins, and DNA, which triggers a self-propagating inflammatory cascade of degenerative events. Dysfunctional mitochondria under oxidative stress conditions are considered a key mediator in progressive neurodegeneration. Exogenous delivery of antioxidants holds promise to alleviate oxidative stress to regain the redox balance. In this regard, natural and synthetic antioxidants have been evaluated. Despite promising results in preclinical studies, clinical translation of antioxidants as a therapy to treat neurodegenerative diseases remains elusive. The issues could be their low bioavailability, instability, limited transport to the target tissue, and/or poor antioxidant capacity, requiring repeated and high dosing, which cannot be administered to humans because of dose-limiting toxicity. Our laboratory is investigating nanoparticle-mediated delivery of antioxidant enzymes to address some of the above issues. Apart from being endogenous, the main advantage of antioxidant enzymes is their catalytic mechanism of action; hence, they are significantly more effective at lower doses in detoxifying the deleterious effects of free radicals than nonenzymatic antioxidants. This review provides a comprehensive analysis of the potential of antioxidant therapy, challenges in their clinical translation, and the role nanoparticles/drug delivery systems could play in addressing these challenges.

Nanoparticles with antioxidant enzymes protect injured spinal cord from neuronal cell apoptosis by attenuating mitochondrial dysfunction.

In spinal cord injury (SCI), the initial damage leads to a rapidly escalating cascade of degenerative events, known as secondary injury. Loss of mitochondrial homeostasis after SCI, mediated primarily by oxidative stress, is considered to play a crucial role in the proliferation of secondary injury cascade. We hypothesized that effective exogenous delivery of antioxidant enzymes - superoxide dismutase (SOD) and catalase (CAT), encapsulated in biodegradable nanoparticles (nano-SOD/CAT) - at the lesion site would protect mitochondria from oxidative stress, and hence the spinal cord from secondary injury. Previously, in a rat contusion model of severe SCI, we demonstrated extravasation and retention of intravenously administered nanoparticles specifically at the lesion site. To test our hypothesis, a single dose of nano-SOD/CAT in saline was administered intravenously 6 h post-injury, and the spinal cords were analyzed one week post-treatment. Mitochondria isolated from the affected region of the spinal cord of nano-SOD/CAT-treated animals demonstrated significantly reduced mitochondrial reactive oxygen species (ROS) activities, increased mitochondrial membrane potential, reduced calcium levels, and also higher adenosine triphosphate (ATP) production capacity than those isolated from the spinal cords of untreated control or SOD/CAT solution treated animals. Although the treatment did not achieve the same mitochondrial function as in the spinal cords of sham control animals, it significantly attenuated mitochondrial dysfunction following SCI. Further, immunohistochemical analyses of the spinal cords of treated animals showed significantly lower ROS, cleaved caspase-3, and cytochrome c activities, leading to reduced spinal cord neuronal cell apoptosis and smaller lesion area than in untreated animals. These results imply that the treatment significantly attenuated progression of secondary injury that was also reflected from less weight loss and improved locomotive recovery of treated vs. untreated animals. In conclusion, nano-SOD/CAT mitigated activation of cascade of degenerating factors by protecting mitochondria and hence the spinal cord from secondary injury. An effective treatment during the acute phase following SCI could potentially have a positive long-term impact on neurological and functional recovery.

Increased HIF-1α and HIF-2α accumulation, but decreased microvascular density, in chronic hyperoxia and hypercapnia in the mouse cerebral cortex.

The partial pressure of oxygen in the brain parenchyma is tightly controlled, and normal brain function is delicately sensitive to continuous and controlled oxygen delivery. The objective of this study was to determine brain angiogenic adaptive changes during chronic normobaric hyperoxia and hypercapnia in mice. Four-month-old C56BL/6 J mice were kept in a normobaric chamber at 50 % O2 and 2.5 % CO2 for up to 3 weeks. Normoxic littermates were kept adjacent to the chamber and maintained on the same schedule. Physiological variables were measured at time points throughout the 3 weeks or when the mice were sacrificed. Freshly collected or fixed brain specimens were analyzed by Western blot analysis and immunohistochemistry (IHC). We found significant accumulation of hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) and increased expression of erythropoietin (EPO), cyclooxygenase-2 (COX-2), and angiopoietin-2 (Ang-2) in hyperoxia and hypercapnia. Conversely, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (KDR/Flk-1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), and prolyl hydroxylase-2 (PHD-2) expressions were decreased in hyperoxia and hypercapnia. Capillary density was significantly diminished by the end of the 3rd week of hyperoxia and hypercapnia as compared to control. We conclude that HIF-independent mechanisms contribute to brain capillary density modulation that is continuously adjusted in accordance with tissue oxygen tension.

Ketosis proportionately spares glucose utilization in brain.

The brain is dependent on glucose as a primary energy substrate, but is capable of utilizing ketones such as ß-hydroxybutyrate and acetoacetate, as occurs with fasting, starvation, or chronic feeding of a ketogenic diet. The relationship between changes in cerebral metabolic rates of glucose (CMRglc) and degree or duration of ketosis remains uncertain. To investigate if CMRglc decreases with chronic ketosis, 2-[(18)F]fluoro-2-deoxy-D-glucose in combination with positron emission tomography, was applied in anesthetized young adult rats fed 3 weeks of either standard or ketogenic diets. Cerebral metabolic rates of glucose (µmol/min per 100 g) was determined in the cerebral cortex and cerebellum using Gjedde-Patlak analysis. The average CMRglc significantly decreased in the cerebral cortex (23.0±4.9 versus 32.9±4.7) and cerebellum (29.3±8.6 versus 41.2±6.4) with increased plasma ketone bodies in the ketotic rats compared with standard diet group. The reduction of CMRglc in both brain regions correlates linearly by ∼9% for each 1 mmol/L increase of total plasma ketone bodies (0.3 to 6.3 mmol/L). Together with our meta-analysis, these data revealed that the degree and duration of ketosis has a major role in determining the corresponding change in CMRglc with ketosis.

Kruppel-like factor 2 protects against ischemic stroke by regulating endothelial blood brain barrier function.

During an ischemic stroke normal brain endothelial function is perturbed, resulting in blood brain barrier (BBB) breakdown with subsequent infiltration of activated inflammatory blood cells, ultimately leading to neuronal cell death. Kruppel-like factor 2 (KLF2) is regulated by flow, is highly expressed in vascular endothelial cells (ECs), and serves as a key molecular switch regulating endothelial function and promoting vascular health. In this study we sought to determine the role of KLF2 in cerebrovascular function and the pathogenesis of ischemic stroke. Transient middle cerebral artery occlusion was performed in KLF2-deficient (KLF2(-/-)), KLF2 overexpressing (KLF2(tg)), and control mice, and stroke volume was analyzed. BBB function was assessed in vivo by real-time neuroimaging using positron emission tomography and Evan's blue dye assay. KLF2(-/-) mice exhibited significantly larger strokes and impairment in BBB function. In contrast, KLF2(tg) mice were protected against ischemic stroke and demonstrated preserved BBB function. In concordance, gain- and loss-of-function studies in primary brain microvascular ECs using transwell assays revealed KLF2 to be BBB protective. Mechanistically, KLF2 was demonstrated, both in vitro and in vivo, to regulate the critical BBB tight junction factor occludin. These data are first to identify endothelial KLF2 as a key regulator of the BBB and a novel neuroprotective factor in ischemic stroke.

Contribution of brain glucose and ketone bodies to oxidative metabolism.

UNLABELLED: Ketone bodies are an alternative energy substrate to glucose in brain. Under conditions of oxidative stress, we hypothesize that ketosis stabilizes glucose metabolism by partitioning glucose away from oxidative metabolism towards ketone body oxidation. In this study we assessed oxidative metabolism in ketotic rat brain using stable isotope mass spectrometry analysis. The contribution of glucose and ketone bodies to oxidative metabolism was studied in cortical brain homogenates isolated from anesthetized ketotic rats. To induce chronic ketosis, rats were fed either a ketogenic (high-fat, carbohydrate restricted) or standard rodent chow for 3 weeks and then infused intravenously with tracers of [U-(13)C] glucose or [U-(13)C] acetoacetate for 60 min. The measured percent contribution of glucose or ketone bodies to oxidative metabolism was analyzed by measuring the (13)C-label incorporation into acetyl-CoA. Using mass spectrometry (gas-chromatography; GC-MS, and liquid-chromatography; LCMS) and isotopomer analysis, the fractional amount of substrate oxidation was measured as the M + 2 enrichment (%) of acetyl-CoA relative to the achieved enrichment of the infused precursors, [U-(13)C]glucose or [U-(13)C] acetoacetate. RESULTS: the percent contribution of glucose oxidation in cortical brain in rats fed the ketogenic diet was 71.2 ± 16.8 (mean% ± SD) compared to the standard chow, 89.0 ± 14.6. Acetoacetate oxidation was significantly higher with ketosis compared to standard chow, 41.7 ± 9.4 vs. 21.9 ± 10.6. These data confer the high oxidative capacity for glucose irrespective of ketotic or non-ketotic states. With ketosis induced by 3 weeks of diet, cortical brain utilizes twice as much acetoacetate compared to non-ketosis.

Decreased VEGF expression and microvascular density, but increased HIF-1 and 2α accumulation and EPO expression in chronic moderate hyperoxia in the mouse brain.

Normal brain function is dependent on continuous and controlled oxygen delivery. Chronic moderate hypoxia leads to angiogenesis, suggesting a modulatory role for oxygen in determining capillary density. The objective of this study was to determine physiologic and brain angiogenic adaptational changes during chronic moderate normobaric hyperoxia in mice. Four-month old C56BL/6J mice were kept in a normobaric chamber at 50% O(2) for up to 3 weeks. Normoxic littermates were kept in the same room outside the chamber. Freshly collected or fixed brain specimens were analyzed by RT-PCR, Western blot analysis and immunohistochemistry. Results show accumulation of hypoxia inducible factors 1 and 2α (HIF-1 and 2α), and increased expression of erythropoietin (EPO), cyclooxygenase-2 (COX-2) and angiopoietin-2 (Ang-2). Conversely, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (KDR/Flk-1), Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and prolylhydroxylase-2 (PHD-2) expressions were decreased. VEGF mRNA level was diminished but there was no change in HIF-1α mRNA and von Hippel Lindau E3 ubiquitin ligase (VHL) protein expression. Microvascular density was significantly diminished by the end of the 3rd week of hyperoxia. Overall, our results are: (1) increased expression of the potent neuroprotective molecule, EPO; (2) diminished expression of the potent angiogenic factor, VEGF; and (3) decreased microvascular density. We can, therefore, conclude that brain microvascular density can be controlled by HIF-independent mechanisms, and that brain capillary density is a continuously adjusted variable with tissue oxygen availability as one of the controlling modulators.

Improvement of neurological recovery and stimulation of neural progenitor cell proliferation by intrathecal administration of Sonic hedgehog.

OBJECT: Sonic hedgehog (Shh) is a glycoprotein molecule that has been shown to be associated with the proliferative capacity of endogenous neural precursor cells during embryonic development. It has also been shown to regulate the proliferative capacity of neural stem cells in the adult subventricular zone (SVZ), which are also upregulated in animal models of ischemic stroke. In the present study, the effects of exogenous administration of intrathecal Shh protein were examined in the setting of a rodent model of ischemic stroke, with particular attention given to endogenous neural stem cell proliferation and migration as well as inducible differences in behavioral recovery. METHODS: A rodent model of ischemic stroke was created using the intraluminal suture method of reversible middle cerebral artery occlusion. Animals were treated with intrathecal administration of Shh protein at 24 hours after the onset of the stroke. Behavioral testing was performed, and the animals were killed for measurements of infarct volume 7 days after stroke. Immunohistochemical staining was performed and measurements of cellular proliferation were obtained, with a focus on the proportion and distribution of neural progenitor cells in the SVZ. These values were compared across experimental groups. RESULTS: Treatment with intrathecal Shh protein resulted in significant improvement in behavioral function compared with the control group, with a significant reduction of ischemic tissue in the cerebral hemisphere. An increase of nestin immunoreactive cells was observed along the SVZ. CONCLUSIONS: Intrathecal Shh agonist at doses that upregulate spinal cord GLI1 transcription increases the population of neural precursor cells after spinal cord injury in adult rats. Intrathecal administration of Shh protein appears to have a neuroprotective effect in animal models of ischemic stroke and is associated with improved behavioral recovery, which may be related to its effects on neurogenesis in the SVZ and could be associated with improved functional recovery.