CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain.

The postnatal forebrain subventricular zone (SVZ) harbors stem cells that give rise to olfactory bulb interneurons throughout life. The identity of stem cells in the adult SVZ has been extensively debated. Although, ependymal cells were once suggested to have stem cell characteristics, subsequent studies have challenged the initial report and postulated that subependymal GFAP(+) cells were the stem cells. Here, we report that, in the adult mouse forebrain, immunoreactivity for a neural stem cell marker, prominin-1/CD133, is exclusively localized to the ependyma, although not all ependymal cells are CD133(+). Using transplantation and genetic lineage tracing approaches, we demonstrate that CD133(+) ependymal cells continuously produce new neurons destined to olfactory bulb. Collectively, our data indicate that, compared with GFAP expressing adult neural stem cells, CD133(+) ependymal cells represent an additional-perhaps more quiescent-stem cell population in the mammalian forebrain.

Gestational hypoxia induces white matter damage in neonatal rats: a new model of periventricular leukomalacia.

In the premature infant, periventricular leukomalacia, usually related to hypoxicischemic white matter damage, is the main cause of neurological impairment. We hypothesized that protracted prenatal hypoxia might induce white matter damage during the perinatal period. Pregnant Sprague-Dawley rats were placed in a chamber supplied with hypoxic gas (10% O2-90% N2) from embryonic day 5 (E5) to E20. Neonatal rat brains were investigated by histology, immunocytochemistry, western blotting, in situ hybridization, DNA fragmentation analysis, and in vivo magnetic resonance imaging (MRI). Body weight of pups subjected to prenatal hypoxia was 10 to 30% lower from P0 to P14 than in controls. Specific white matter cysts were detected between P0 and P7 in pups subjected to prenatal hypoxia, in addition to abnormal extra-cellular matrix, increased lipid peroxidation, white matter cell death detected by TUNEL, and increased activated macrophage counts in white matter. Subsequently, gliotic scars and delayed myelination primarily involving immature oligodendrocytes were seen. In vivo MRI with T1, T2, and diffusion sequences disclosed similar findings immediately after birth, showing strong correlations with histological abnormalities. We speculate that protracted prenatal hypoxia in rat induces white matter damage occurring through local inflammatory response and oxidative stress linked to re-oxygenation during the perinatal period.

Neural progenitors populate the cerebrospinal fluid of preterm patients with hydrocephalus.

OBJECTIVES: To evaluate cerebrospinal fluid (CSF) of preterm patients with hydrocephalus for neural progenitors. STUDY DESIGN: This report describes a prospective study of CSF obtained from preterm infants, either with progressive posthemorrhagic hydrocephalus (PPHH) or without known intercranial pathology. Cells recovered by centrifugation were analyzed by reverse transcriptase-polymerase chain reaction or by immunocytometry. Alternatively, cells were cultured by using methods permissive to neural progenitor growth and analyzed by immunocytochemistry and Western blotting. RESULTS: Human CSF cells were obtained from 20 preterm infants at approximately 27 weeks estimated gestational age (15 infants with PPHH, 5 control infants). The number of these cells removed over time from patients with PPHH were substantial, based on our calculations. Cells recovered from patients with PPHH transcribe markers for neural progenitors, all the mature cells types of the central nervous system, and a large battery of chondroitin sulfate proteoglycan genes, including the entire aggrecan/lectican family. These cells proliferated in culture, and precursor markers were detected by Western blotting, immunocytochemistry, and cytometry. Cells could not be cultured from control patients. CONCLUSIONS: Neural progenitor accumulation in CSF could confound the clinical interpretation of CSF cell counts in hydrocephalus and may play as yet undetermined roles in the biology of injury after hydrocephalus. These findings suggest the potential for neural stem cell propagation from CSF.

Accumulation of transforming growth factor-beta2 and nitrated chondroitin sulfate proteoglycans in cerebrospinal fluid correlates with poor neurologic outcome in preterm hydrocephalus.

BACKGROUND: Progressive post-hemorrhagic hydrocephalus in preterm infants strongly predicts abnormal neurologic development, and often accompanies cystic periventricular leukomalacia (cPVL). Transforming growth factor-beta1 (TGF-beta1), associated with hydrocephalus, can upregulate the chondroitin sulfate proteoglycan (CSPG) synthesis. To date, CSPG and their nitrated metabolites (NT-CSPG) have not been evaluated in hydrocephalus. OBJECTIVES: We hypothesized that TGF-beta1, TGF-beta2, CSPG, and NT-CSPG would accumulate in cerebrospinal fluid (CSF) in preterm hydrocephalus, and their concentrations would correlate with poor long-term outcomes. METHODS: TGF-beta1, TGF-beta2, CSPG, and NT-CSPG concentrations in CSF were measured prospectively by ELISA in 29 preterm newborns with (n=22) or without (n=34) progressive post-hemorrhagic hydrocephalus, and correlated with progressive neonatal hydrocephalus and neurologic outcome. Only concentrations from each patient's initial CSF sample were used for statistical analysis. RESULTS: Compared to neonates without hydrocephalus, CSF [TGF-beta1], [TGF-beta2], [CSPG] and [NT-CSPG] were significantly greater by >3-, >35-, >8-, and >3-fold, respectively. Unlike CSF [TGF-beta2] and [CSPG], [TGF-beta1] correlated with CSF [total protein]. Only CSF [NT-CSPG] correlated with cPVL. Unlike [TGF-beta2] or [CSPG], [NT-CSPG] correlation with preterm progressive post-hemorrhagic hydrocephalus (PPHH) was explained entirely by the presence of cPVL among these patients. [TGF-beta2] was >20-fold greater in preterm survivors who required a ventriculoperitoneal shunt for PPHH (n=9), as compared to survivors who did not require a shunt (n=2), or those without hydrocephalus (n=12). [TGF-beta2] and [NT-CSPG] correlated inversely with Bayley Index Scores (15.0 months median adjusted age). CONCLUSIONS: This is the first report that [TGF-beta2], [CSPG], and [NT-CSPG], measured well before term, accumulate abnormally in preterm progressive post-hemorrhagic hydrocephalus CSF, and correlate with adverse neurologic outcome.

Use of a novel double-sandwich enzyme-linked immunosorbent assay method for assaying chondroitin sulfate proteoglycans that bear 3-nitrotyrosine core protein modifications, a previously unrecognized proteoglycan modification in hydrocephalus.

3-Nitrotyrosine is a useful marker for nitric oxide-mediated tissue injury. However, which proteins are preferred peroxynitrite modification targets is unclear. Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid of human neonates with hydrocephalus and may be a target for peroxynitrite modification. We examined (1). whether CSPG core protein can be modified by peroxynitrite in vitro; (2). to what degree in comparison to bovine serum albumin (BSA), the most commonly used nitrated protein standard; (3). whether nitrated CSPGs can be measured directly in biological samples; and (4). whether nitrated proteoglycan concentrations in cerebrospinal fluid correlate with disease. In vitro nitration of bovine aggrecan was performed by exposure to different peroxynitrite concentrations, and 3-nitrotyrosine products were measured. Bovine serum albumin (BSA) nitration was also performed in comparison. A larger percentage of tyrosine residues were nitrated in aggrecan than in BSA under all conditions tested. An enzyme-linked immunosorbent assay (ELISA) for 3-nitrotyrosine consistently overestimated aggrecan nitration when nitrated BSA was used as the standard. This is important as most current assays of nitration in biological samples use nitrated BSA as the standard. Therefore, if nitrated CPSGs were a substantial portion of the nitrated proteins in a sample, total nitrated protein content would be overestimated. Aggrecan retained its function of binding hyaluronic acid despite substantial nitration. A double-sandwich ELISA was developed for nitrated CSPGs in biological samples, using nitrated aggrecan as standard. [Nitrated CSPG] was found to be significantly elevated in preterm hydrocephalus cerebrospinal fluid (P<0.02), but correlated poorly with cerebrospinal fluid [nitric oxide] (P>0.069), suggesting that nitrated CSPG and NO levels may be independant markers of tissue injury. Peroxynitrite-mediated protein tyrosine nitration is a previously unrecognized modification of CSPGs, and may reflect level of brain injury in hydrocephalus.

Hemodynamic changes in anemic premature infants: are we allowing the hematocrits to fall too low?

OBJECTIVE: Currently, many nurseries allow hematocrits to fall to <21% in apparently "stable" premature infants before considering a blood transfusion. We evaluated clinical changes and hemodynamic changes by echocardiogram in "stable" anemic premature infants before, during, and after transfusion. METHODS: "Stable" premature infants (< or =32 weeks' gestation) who were to receive transfusions (2 aliquots of 10 mL/kg packed red blood cells, 12 hours apart) were eligible for prospective enrollment. Cardiac function by echocardiography and vital signs were measured 4 times: 1 to 3 hours before and 2 to 4 hours after the initial aliquot and 4 to 7 hours and 27 to 34 hours after the second aliquot. Infants were grouped prospectively according to pretransfusion hematocrit ranges for analysis: < or =21% (low), 22% to 26% (mid), and > or =27% (high). RESULTS: Thirty-two infants were enrolled. No differences were observed between the groups in sex, birth weight, postconceptional age, or postnatal weight at enrollment. Before transfusion, low- and mid-range groups had higher left ventricular end systolic and diastolic diameters, in comparison with high range. Low range had increased stroke volume in comparison with the high-range group. These changes persisted after transfusion. Mean diastolic blood pressure rose and peak velocity in the aorta fell in the low-range group after transfusion. Pretransfusion hematocrit was correlated with but poorly predictive of echocardiographic measurements. Infants with inappropriate weight gain had increased ventricular end diastolic diameters, consistent with congestive heart failure. CONCLUSIONS: Apparently "stable" anemic premature infants may be in a clinically unrecognized high cardiac output state, and some echocardiographic measurements do not improve within 48 hours after transfusion. The benefits of transfusion practices guided by measures of cardiac function should be evaluated.

Neural precursors express multiple chondroitin sulfate proteoglycans, including the lectican family.

Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid (CSF) of both human neonates with preterm hydrocephalus, and P8 hydrocephalic mice. We hypothesized CSF CSPGs are synthesized by neural precursors, separated from ventricular CSF by ependyma, which is often disrupted in hydrocephalus. Western blotting demonstrates that neural precursors cultured as neurospheres secrete CSPGs (> 30 microg/ml) into their media which appear to be very similar to these CSF CSPGs. Some CSPGs bear the stage-specific embryonic antigen-1 (ssea-1), associated with embryonic/neural stem cells. Neurospheres transcribe many CSPG genes, including the entire aggrecan/lectican family, phosphacan, and tenascin. Phosphacan can be detected in media by Western blotting. Aggrecan can be detected in media after purification using hyaluronic acid affinity chromatography. During differentiation, neurospheres downregulate CSPGs. This is the first report to show that proliferating neural precursors synthesize lecticans, including aggrecan, which are downregulated with differentiation. These observations suggest novel links between CSPGs and CNS precursor biology.