High phenylalanine concentrations induce demyelination and microglial activation in mouse cerebellar organotypic slices.

Phenylketonuria (PKU) is an inborn error of metabolism. Mutations in the enzyme phenylalanine hydroxylase (PAH)-encoding gene lead to a decreased metabolism of the amino acid phenylalanine (Phe). The deficiency in PAH increases Phe levels in blood and brain. Accumulation of Phe can lead to delayed development, psychiatric problems and cognitive impairment. White matter (WM) damage is a neuropathological hallmark of PKU and can be seen even in early detected and treated PKU patients. The mechanisms linking high Phe concentrations to WM abnormalities remain unclear. We tested the effects of high Phe concentrations on myelin in three in vitro models of increasing complexity: two simple cell culture models and one model that preserves local brain tissue architecture, a cerebellar organotypic slice culture prepared from postnatal day (P) 8 CD-1 mice. Various Phe concentrations (0.1-10 mM) and durations of exposure were tested. We found no toxic effect of high Phe in the cell culture models. On the contrary, the treatment promoted the maturation of oligodendrocytes, particularly at the highest, non-physiological Phe concentrations. Exposure of cerebellar organotypic slices to 2.4 mM Phe for 21 days in vitro (DIV), but not 7 or 10 DIV, resulted in a significant decrease in myelin basic protein (MBP), calbindin-stained neurites, and neurites co-stained with MBP. Following exposure to a toxic concentration of Phe, a switch to the control medium for 7 days did not lead to remyelination, while very active remyelination was seen in slices following demyelination with lysolecithin. An enhanced number of microglia, displaying an activated type morphology, was seen after exposure of the slices to 2.4 mM Phe for 10 or 21 DIV. The results suggest that prolonged exposure to high Phe concentrations can induce microglial activation preceding significant disruption of myelin.

A new ketogenic formulation improves functional outcome and reduces tissue loss following traumatic brain injury in adult mice.

Rationale: Traumatic brain injury (TBI) leads to neurological impairment, with no satisfactory treatments available. Classical ketogenic diets (KD), which reduce reliance on carbohydrates and provide ketones as fuel, have neuroprotective potential, but their high fat content reduces compliance, and experimental evidence suggests they protect juvenile brain against TBI, but not adult brain, which would strongly limit their applicability in TBI. Methods: We designed a new-KD with a fat to carbohydrate plus protein ratio of 2:1, containing medium chain triglycerides (MCT), docosahexaenoic acid (DHA), low glycaemic index carbohydrates, fibres and the ketogenic amino acid leucine, and evaluated its neuroprotective potential in adult TBI. Adult male C57BL6 mice were injured by controlled cortical impact (CCI) and assessed for 70 days, during which they received a control diet or the new-KD. Results: The new-KD, that markedly increased plasma Beta-hydroxybutyrate (ß-HB), significantly attenuated sensorimotor deficits and corrected spatial memory deficit. The lesion size, perilesional inflammation and oxidation were markedly reduced. Oligodendrocyte loss appeared to be significantly reduced. TBI activated the mTOR pathway and the new-KD enhanced this increase and increased histone acetylation and methylation. Conclusion: The behavioural improvement and tissue protection provide proof of principle that this new formulation has therapeutic potential in adult TBI.

A Single Injection of Docosahexaenoic Acid Induces a Pro-Resolving Lipid Mediator Profile in the Injured Tissue and a Long-Lasting Reduction in Neurological Deficit after Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) can lead to life-changing neurological deficits, which reflect the fast-evolving secondary injury post-trauma. There is a need for acute protective interventions, and the aim of this study was to explore in an experimental TBI model the neuroprotective potential of a single bolus of a neuroactive omega-3 fatty acid, docosahexaenoic acid (DHA), administered in a time window feasible for emergency services. Adult mice received a controlled cortical impact injury (CCI) and neurological impairment was assessed with the modified Neurological Severity Score (mNSS) up to 28 days post-injury. DHA (500 nmol/kg) or saline were injected intravenously at 30 min post-injury. The lipid mediator profile was assessed in the injured hemisphere at 3 h post-CCI. After completion of behavioral tests and lesion assessment using magnetic resonance imaging, over 7 days or 28 days post-TBI, the tissue was analyzed by immunohistochemistry. The single DHA bolus significantly reduced the injury-induced neurological deficit and increased pro-resolving mediators in the injured brain. DHA significantly reduced lesion size, the microglia and astrocytic reaction, and oxidation, and decreased the accumulation of beta-amyloid precursor protein (APP), indicating a reduced axonal injury at 7 days post-TBI. DHA reduced the neurofilament light levels in plasma at 28 days. Therefore, an acute single bolus of DHA post-TBI, in a time window relevant for acute emergency intervention, can induce a long-lasting and significant improvement in neurological outcome, and this is accompanied by a marked upregulation of neuroprotective mediators, including the DHA-derived resolvins and protectins.

Brain Phospholipid Precursors Administered Post-Injury Reduce Tissue Damage and Improve Neurological Outcome in Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) leads to cellular loss, destabilization of membranes, disruption of synapses and altered brain connectivity, and increased risk of neurodegenerative disease. A significant and long-lasting decrease in phospholipids (PLs), essential membrane constituents, has recently been reported in plasma and brain tissue, in human and experimental TBI. We hypothesized that supporting PL synthesis post-injury could improve outcome post-TBI. We tested this hypothesis using a multi-nutrient combination designed to support the biosynthesis of PLs and available for clinical use. The multi-nutrient, Fortasyn® Connect (FC), contains polyunsaturated omega-3 fatty acids, choline, uridine, vitamins, cofactors required for PL biosynthesis, and has been shown to have significant beneficial effects in early Alzheimer's disease. Male C57BL/6 mice received a controlled cortical impact injury and then were fed a control diet or a diet enriched with FC for 70 days. FC led to a significantly improved sensorimotor outcome and cognition, reduced lesion size and oligodendrocyte loss, and it restored myelin. It reversed the loss of the synaptic protein synaptophysin and decreased levels of the axon growth inhibitor, Nogo-A, thus creating a permissive environment. It decreased microglia activation and the rise in ß-amyloid precursor protein and restored the depressed neurogenesis. The effects of this medical multi-nutrient suggest that support of PL biosynthesis post-TBI, a new treatment paradigm, has significant therapeutic potential in this neurological condition for which there is no satisfactory treatment. The multi-nutrient tested has been used in dementia patients and is safe and well tolerated, which would enable rapid clinical exploration in TBI.

Combination of vascular endothelial and fibroblast growth factor 2 for induction of neurogenesis and angiogenesis after traumatic brain injury.

Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) are potent mitogens for endogenous neural stem cells (eNSC) and also induce angiogenesis. We infused the individual factors or their combination into the lateral ventricles of mice for 7 days after traumatic brain injury (TBI) in order to evaluate the effects on functional outcome and on eNSC proliferation and differentiation. The results show that VEGF induced a significant increment in the number of proliferating eNSC in the subventricular zone and in the perilesion cortex and that combination of FGF2 and VEGF did not augment the effects of VEGF alone. Fate analysis showed that most newborn cells differentiated into astrocytes and oligodendroglia while only a few cells differentiated into neurons. Functional outcome was significantly better in mice treated with VEGF, FGF2, or their combination as compared to vehicle. Injury size was significantly reduced only in mice treated with VEGF suggesting additional neuroprotective effects for VEGF. Combination therapy did not have an additive effect on outcome or neuronal differentiation. In conclusion, FGF2-VEGF combination does not augment neurogenesis and angiogenesis or reduce lesion volumes after TBI compared with individual factors. This may suggest the existence of a ceiling effect for brain regeneration.

The anti-inflammatory drug carprofen improves long-term outcome and induces gliogenesis after traumatic brain injury.

Traumatic brain injury (TBI) initiates acute and chronic inflammatory processes involving cyclooxygenase-2 (COX-2), which may have detrimental effects on outcome and especially on brain regeneration. Therefore we aimed to study whether carprofen, a COX-2 inhibitor, would improve outcome and increase neurogenesis after TBI. TBI was induced in Sabra mice that were then treated with vehicle or carprofen for 7 days. Functional outcome was evaluated with the Neurological Severity Score (NSS).Cytokine levels were assessed 4 h post-TBI and water content was measured 24 h post TBI. Mice were given BrdU to label newborn cells for 10 days. The animals were killed 90 days post-TBI and the lesion size as well as newborn cell fate were assessed. Carprofen significantly reduced lesion size (p=0.002), decreased water content in the lesioned cortex (p=0.03), reduced the number of microglia in the lesioned cortex (p<0.0001), and lowered the levels of proinflammatory cytokines (IL-1ß, p=0.03; IL-6, p=0.02). Carprofen led to significantly larger improvements in functional outcome (p≤0.008) which were durable over 90 days. Carprofen also induced a threefold increase in the proliferation of new cells in the peri-lesion area (p≤0.002), but newborn cells differentiated mainly into glia in both groups. Carprofen is neuroprotective and induces cell proliferation and gliogenesis after TBI. Treatment with carprofen is consistently associated with better functional outcome. Our results imply that anti-inflammatory drugs may represent novel therapeutic options for TBI.

Vascular endothelial growth factor increases neurogenesis after traumatic brain injury.

Activation of endogenous stem cells has been proposed as a novel form of therapy in a variety of neurologic disorders including traumatic brain injury (TBI). Vascular endothelial growth factor (VEGF) is expressed in the brain after TBI and serves as a potent activator of angiogenesis and neurogenesis. In this study, we infused exogenous VEGF into the lateral ventricles of mice for 7 days after TBI using mini-osmotic pumps to evaluate the effects on recovery and functional outcome. The results of our study show that VEGF significantly increases the number of proliferating cells in the subventricular zone and in the perilesion cortex. Fate analysis showed that most newborn cells differentiated into astrocytes and oligodendroglia and only a few cells differentiated into neurons. Functional outcome was significantly better in mice treated with VEGF compared with vehicle-treated animals after TBI. Injury size was significantly smaller at 90 days after TBI in VEGF-treated animals, suggesting additional neuroprotective effects of VEGF. In conclusion, VEGF significantly augments neurogenesis and angiogenesis and reduces lesion volumes after TBI. These changes are associated with significant improvement in recovery rates and functional outcome.