Secondary hypoxia is a known contributor to adverse outcomes in patients with traumatic brain injury (TBI). Based on the evidence that hypoxia and TBI in isolation induce neuroinflammation, we investigated whether TBI combined with hypoxia enhances cerebral cytokine production. We also explored whether increased concentrations of injury biomarkers discriminate between hypoxic (Hx) and normoxic (Nx) patients, correlate to worse outcome, and depend on blood-brain barrier (BBB) dysfunction. Forty-two TBI patients with Glasgow Coma Scale ≤8 were recruited. Cerebrospinal fluid (CSF) and serum were collected over 6 days. Patients were divided into Hx (n=22) and Nx (n=20) groups. Eight cytokines were measured in the CSF; albumin, S100, myelin basic protein (MBP) and neuronal specific enolase (NSE) were quantified in serum. CSF/serum albumin quotient was calculated for BBB function. Glasgow Outcome Scale Extended (GOSE) was assessed at 6 months post-TBI. Production of granulocye macrophage-colony stimulating factor (GM-CSF) was higher, and profiles of GM-CSF, interferon (IFN)-γ and, to a lesser extent, tumor necrosis factor (TNF), were prolonged in the CSF of Hx but not Nx patients at 4-5 days post-TBI. Interleukin (IL)-2, IL-4, IL-6, and IL-10 increased similarly in both Hx and Nx groups. S100, MBP, and NSE were significantly higher in Hx patients with unfavorable outcome. Among these three biomarkers, S100 showed the strongest correlations to GOSE after TBI-Hx. Elevated CSF/serum albumin quotients lasted for 5 days post-TBI and displayed similar profiles in Hx and Nx patients. We demonstrate for the first time that post-TBI hypoxia is associated with prolonged neuroinflammation, amplified extravasation of biomarkers, and poor outcome. S100 and MBP could be implemented to track the occurrence of post-TBI hypoxia, and prompt adequate treatment.

Pregnane steroids have sedative and neuroprotective effects on the brain, due to interactions with the steroid-binding site of the GABA A receptor. In the adult brain, synthesis of the pregnane steroids is increased in response to stress. Therefore, we have used umbilicoplacental embolization to mimic chronic placental insufficiency during late gestation in sheep, to investigate the expression of the steroidogenic enzymes P450scc, 5␣-reductase type I (5␣RI), 5␣-reductase type II (5␣RII), and allopregnanolone (AP) content in the fetal brain. Umbilicoplacental embolization was induced from 114 d gestation (term~147 d) by daily injection of inert microspheres into the umbilical artery and continued for 17-23 d. Fetal arterial oxygen saturation was reduced to~60% of the preembolization value in each fetus, with a significant reduction in blood arterial PO 2 , pH, and plasma glucose concentrations (p Ͻ 0.05) and a significant increase in blood arterial PCO 2 and plasma lactate concentrations (p Ͻ 0.05). At postmortem at 131-137 d gestation, embolized fetuses were growth-restricted (2.10 Ϯ 0.14 kg, n ϭ 5) compared with agematched controls (4.43 Ϯ 0.56 kg, n ϭ 7, p Ͻ 0.05). Umbilicoplacental embolized fetuses showed increased P450scc expression in the primary motor cortex; 5␣RI expression was not changed in any of the regions examined, whereas 5␣RII expression was markedly increased in all brain regions. Brain AP content did not significantly change, whereas plasma concentrations were increased. These findings suggest that the increased expression of P450scc and 5␣RII may be a response that maintains AP concentration in the fetal brain after compromised placental function and/or intrauterine stress. Chronic hypoxemia and placental insufficiency are associated with fetal growth retardation (1). Generally, the brains of these growth-retarded fetuses are either not smaller, or are reduced to a lesser degree compared with the overall size of the fetus, suggesting that the brain is able to adapt to the intrauterine conditions that compromise fetal growth. On the other hand, growth-retarded and premature infants are at a greater risk of perinatal brain damage (2), indicating that some of these adaptive changes leave the brain more vulnerable to cytotoxic damage arising from a range of insults, such as acute hypoxia, asphyxia, or infection-induced inflammation.Pregnane steroids, including AP, modify the excitability of the CNS by interaction with the GABA A receptor at the steroid binding site. In the adult, AP has been shown to have potent anxiolytic (3, 4), anticonvulsant (5, 6), sedative/hypnotic, and anesthetic effects (7) on behavior. A constitutive role for neuroactive steroids in the developing brain was proposed after the observation that progesterone metabolites such as AP appear to maintain the low level of arousal-like behavior that typifies fetal life (8,9). In the rat, stressful stimuli of handling or swim stress have been shown to increase plasma AP content (10, 11), suggesting that modulation of the GABA A rece...

Pregnane steroids have sedative and neuroprotective effects on the brain as a result of interactions with the steroid-binding site of the GABA A receptor. To determine whether the fetal brain is able to synthesize pregnane steroids de novo from cholesterol, we measured the expression of cytochrome P450 side-chain cleavage (P450scc) and 5␣-reductase type II (5␣RII) enzymes in fetal sheep from 72 to 144 d gestation (term~147 d) and in newborn lambs at 3 and 19-26 d of age. Both P450scc and 5␣RII expression was detectable by 90 d gestation in the major regions of the brain and also in the adrenal glands. Expression increased with advancing gestation and was either maintained at fetal levels or increased further after birth. In contrast, the relatively high content (200-400 pmol/g) of allopregnanolone (5␣-pregnan-3␣-ol-20-one), a major sedative 5␣-pregnane steroid, present throughout the brain from 90 d gestation to term, was reduced significantly (Ͻ50 pmol/g) immediately after birth. These results suggest that although the perinatal brain has the enzymes potentially to synthesize pregnane steroids de novo from cholesterol, either the placenta is a major source of these steroids to the brain or other factors associated with intrauterine life may be responsible for high levels of allopregnanolone production in the fetal brain until birth. Abbreviations AP, allopregnanolone, 5␣-pregnan-3␣-ol-20-one P450scc, P450 side chain cleavage enzyme 5␣RII, 5␣-reductase type II enzyme PMC, primary motor cortex GABA A ,␥-aminobutyric acid/benzodiazepine receptor 5␣-DHP, 5␣-dihydroprogesterone Neuroactive steroids such as allopregnanolone (AP) are potent neuromodulators that modify the excitability of the CNS by interaction with the ␥-aminobutyric acid/benzodiazepine receptor-chloride ionophore (GABA A receptor). In the adult AP is a positive allosteric modulator of the GABA A receptor with potent anxiolytic (1, 2), anticonvulsant (3, 4), sedative/ hypnotic, and anesthetic effects (5) on behavior. Prenatally, neuroactive steroids have been shown to suppress fetal activity in late gestation and seem to have a role in maintaining the low level of arousal-like behavior that typifies fetal life (6, 7). This interaction between AP and the GABA A receptor complex is responsible for the major pharmacologic actions of AP and is distinct from the genomic effects exerted by other steroids such as progesterone (8). In the brain, neuroactive steroids such as AP are synthesized de novo from cholesterol, but a proportion of the pool of steroids may be derived from precursors in blood that enter the brain across the blood-brain barrier (9 -11). Thus, peripheral steroidogenesis may influence neuroactive steroid content in the brain.The cytochrome P450 side-chain cleavage enzyme (P450scc) catalyzes the irreversible conversion of cholesterol to pregnenolone on the inner side of the mitochondrial membrane (12). The primary site of pregnenolone synthesis in the brain seems to be in oligodendrocytes and astrocytes, with significantly less synthesis occ...

Understanding the kinetics, thermodynamics, and molecular mechanisms of liquid–liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.