Nestin expression in pancreatic stellate cells and angiogenic endothelial cells

Nestin is an intermediate filament protein expressed by neuroepithelial stem cells and which has been proposed to represent also a marker for putative islet stem cells. The aim of this study was to characterize the cell type(s) expressing nestin in the rat pancreas. By immunohistochemistry, nestin positivity was localized exclusively in mesenchymal cells of normal and regenerating adult pancreas. In the latter condition, the number of nestin-positive cells and the intensity of nestin immunoreactivity were greatly increased. Most nestin-positive cells had the morphology of stellate cells, a type of pericyte associated with blood vessels which has been previously reported to occur in liver and pancreas. In addition, nestin positivity was present in endothelial cells from neocapillaries during pancreas regeneration, and in all blood vessels during morphogenesis in fetal pancreas. Nestin expression was not found in the ductal epithelial cells from which islet cells originate in fetal and regenerating pancreas. In primary pancreatic tissue explants, nestin-positive mesenchymal cells rapidly attached to plastic and proliferated. These cells also expressed desmin, vimentin, and glial fibrillary acidic protein which are known to represent stellate cell markers. In summary, nestin in the pancreas is primarily a marker for reactive stellate cells, or pericytes, and endothelial cells during active angiogenesis.     

Focal cerebral ischaemia induces corticotropin releasing factor (CRF) vascular immunoreactivity in rat occluded hemisphere

Corticotropin-releasing factor (CRF) induces the dilatation of cerebral blood vessels and increases cerebral blood flow (CBF). CRF receptor antagonists reduce ischaemic damage in the rat. In the present study, the expression of CRF around cerebral vessels has been investigated in the rat. No CRF immunoreactivity was identified around pial or intracerebral vessels in the absence of cerebral ischaemia. Four hours after middle cerebral artery occlusion (MCAo), intensely CRF-positive blood vessels were evident on the ischaemic cortical surface and in the peri-infarct and infarct zone. Increased CRF immunoreactivity was also detected in swollen axons in subcortical white matter, caudate nucleus and lateral olfactory tract of the ipsilateral hemisphere, consistent with the failure of axonal transport. These data provide morphologic support for a role of CRF in the pathophysiology of cerebral ischaemia.     

Nestin expression in rat adrenal gland

The constituents of the intermediate filament network of adrenal gland cells have not been deeply investigated in vivo. Adrenocortical cells have been reported to express cytokeratins and vimentin, but the intermediate filament components of the adrenomedullary cells are still unknown. Nestin is an intermediate filament protein that is mainly expressed in the developing nervous and muscle systems. It has been reported to be unable to form filaments by itself and it co-assembles with vimentin. Using immunocytochemical and biochemical approaches, the present study demonstrates that nestin is expressed in situ either in the cortex or in the medulla of adult rat adrenal glands. Nestin-negative cells prevalently form the zona glomerulosa whereas the zona fasciculata and the zona reticularis are mainly nestin-immunoreactive. Nestin-positive cells always express vimentin-like immunoreactivity but several cells apparently expressing only vimentin are detectable too. Nestin is also expressed by adrenomedullary cells that also display a faint vimentin-like immunoreactivity. We hypothesise that the inconstant detection of nestin in adrenocortical cells depends on their different functional moments. Moreover, even though our data do not allow to confirm vimentin in adrenomedullary cells, in situ detection of nestin in the adrenal medulla indirectly supports in vivo expression of vimentin in chromaffin cells.     

Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) alpha1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln alpha1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16-19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln alpha1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln alpha1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln alpha1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

The radial glia antibody RC2 recognizes a protein encoded by Nestin

The RC2 antibody is widely used to label mouse radial glial cells in the developing central nervous system. While the antibody is known to recognize a 295-kDa intermediate filament proximal protein, the gene encoding the RC2 antigen remains to be identified. Here, we present evidences clearly demonstrating that Nestin encodes the RC2 antigen. First, the RC2 antigen and nestin have the same molecular weight and very similar tissue distribution. Second, genetic manipulations altering nestin expression also exert the same effect on the expression of the RC2 antigen. In particular, Nestin null mutation completely abolishes the RC2 immunoreactivity. Third, the expression of a truncated mouse nestin in Nestin−/− cells produces a small RC2 antigen whose size is the same to that of the truncated nestin. Furthermore, our data suggest that the RC2 antibody recognizes the C-terminal domain of nestin with unidentified posttranslational modification(s).     

Laminin-like antigen in rat CNS neurons: distribution and changes upon brain injury and nerve growth factor treatment

Using several antibodies against rat or human laminin and an avidin-biotin immunocytochemical protocol, laminin-like immunoreactivity was detectable in the rat nervous system in expected locations, i.e., associated with blood vessels and reactive astrocytes. However, laminin staining was also abundantly present within neuronal cell bodies in most parts of the developing and adult rat CNS. Medial septum neuronal immunoreactivity was lost after septo-hippocampal disconnection, but could be preserved or even restored by intraventricular administration of nerve growth factor. Thus, at least for medial septum neurons, this laminin-like molecule can be accumulated or produced independent of direct hippocampal (target) contact. It remains to be determined whether CNS neuronal "laminin" processes activities similar to those found for laminin in vitro.     

Laminin α1-Chain Shows a Restricted Distribution in Epithelial Basement Membranes of Fetal and Adult Human Tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) α1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln α1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16–19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln α1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln α1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln α1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

Intermediate filament protein nestin is expressed in developing kidney and heart and might be regulated by the Wilms' tumor suppressor Wt1

Nestin is an intermediate filament protein originally described in neural stem cells and a variety of progenitor cells. More recently, nestin was detected in rat kidney podocytes. We show here that nestin is expressed in a developmentally regulated pattern in the kidney. Nestin was detected by immunohistochemistry in the condensing mesenchyme surrounding the ureter, in developing glomeruli, in podocytes of the adult kidney, and in a podocyte cell line. Nestin shared a striking overlap in expression with the Wilms' tumor suppressor Wt1. Nestin was significantly upregulated in a cell line with inducible Wt1 expression upon induction of Wt1. Cotransfection experiments in human embryonic kidney cells (HEK293) revealed stimulation of a nestin intron 2 enhancer element up to six-fold by the Wt1(-KTS) splice variant. Nestin expression was significantly reduced in an inducible mouse model of glomerular disease. This model is based on podocyte-specific overexpression of Pax2 and associated with a loss of Wt1 expression. Furthermore, also in the developing heart, nestin was found in an overlapping pattern with Wt1 in the epicardium and the forming coronary vessels. Strikingly, in the hearts of Wt1 knockout mice, nestin was barely detectable compared with the hearts of wild-type embryos. Our results show that nestin is expressed at different stages of kidney and cardiac development and suggest that its expression in these organs might be regulated by the Wilms' tumor suppressor Wt1.     

The Neural Stem/Progenitor Cell Marker Nestin Is Expressed in Proliferative Endothelial Cells,but Not in Mature Vasculature

Nestin is an intermediate filament protein that is known as a neural stem/progenitor cell marker. It is expressed in undifferentiated central nervous system (CNS) cells during development, but also in normal adult CNS and in CNS tumor cells. Additionally, nestin is expressed in endothelial cells (ECs) of CNS tumor tissues and of adult tissues that replenish by angiogenesis. However, the regulation of nestin expression in vascular endothelium has not been analyzed in detail. This study showed that nestin expression was observed in proliferating endothelial progenitor cells (EPCs), but not in mature ECs. In adherent cultured cells derived from bone marrow cells, EPCs that highly expressed nestin also expressed the endothelial marker CD31 and the proliferation marker Ki67. ECs cultured without growth factors showed attenuated nestin immunoreactivity as they matured. Transgenic mice that carried the enhanced green fluorescent protein under the control of the CNS-specific second intronic enhancer of the nestin gene showed no reporter gene expression in EPCs. This indicated that the mechanisms of nestin gene expression were different in EPCs and CNS cells. Immunohistochemistry showed nestin expression in neovascular cells from two distinct murine models. Our results demonstrate that nestin can be used as a marker protein for neovascularization. (J Histochem Cytochem 58:721–730, 2010)     

Nestin expression in adult and developing human kidney.

Nestin is considered a marker of neurogenic and myogenic precursor cells. Its arrangement is regulated by cyclin-dependent kinase 5 (CDK5), which is expressed in murine podocytes. We investigated nestin expression in human adult and fetal kidney as well as CDK5 presence in adult human podocytes. Confocal microscopy demonstrated that adult glomeruli display nestin immunoreactivity in vimentin-expressing cells with the podocyte morphology and not in cells bearing the endothelial marker CD31. Glomerular nestin-positive cells were CDK5 immunoreactive as well. Western blotting of the intermediate filament-enriched cytoskeletal fraction and coimmunoprecipitation of nestin with anti-CDK5 antibodies confirmed these results. Nestin was also detected in developing glomeruli within immature podocytes and a few other cells. Confocal microscopy of experiments conducted with antibodies against nestin and endothelial markers demonstrated that endothelial cells belonging to capillaries invading the lower cleft of S-shaped bodies and the immature glomeruli were nestin immunoreactive. Similar experiments carried out with antibodies raised against nestin and alpha-smooth muscle actin showed that the first mesangial cells that populate the developing glomeruli expressed nestin. In conclusion, nestin is expressed in the human kidney from the first steps of glomerulogenesis within podocytes, mesangial, and endothelial cells. This expression, restricted to podocytes in mature glomeruli, appears associated with CDK5.     

SPARC/Osteonectin mRNA Is Induced in Blood Vessels Following Injury to the Adult Rat Cerebral Cortex

Recently we described the pattern of expression of the anti-adhesive glycoprotein SPARC/osteonectin in the developing and adult brain. SPARC mRNA was present in developing blood vessels during neurogenesis, but was not detected in the mature vasculature. We have now examined the effect of a lesion to the adult rat cerebral cortex on the expression of SPARC by in situ hybridization. SPARC mRNA was increased in the zone proximal to the wound at 3 to 10 days after cortical brain injury. During this period, SPARC was induced in mature blood vessels close to the lesion site and in blood vessels which develop following injury. These results suggest a role for SPARC in the process of angiogenesis following injury to the adult cerebral cortex.     

Differential expression of BK channel isoforms and beta-subunits in rat neuro-vascular tissues

We investigated the expression of splice variants and beta-subunits of the BK channel (big conductance Ca2+-activated K+ channel, Slo1, MaxiK, KCa1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An alpha-subunit splice variant X1(+24) was found expressed (RT-PCR) in nervous tissue only where also the SS4(+81) variant was dominating with little expression of the short form SS4(0). SS4(+81) was present in some cerebral vessels too. The SS2(+174) variant (STREX) was found in both blood vessels and in nervous tissue. In situ hybridization data supported the finding of SS4(+81) and SS2(+174) in vascular smooth muscle and trigeminal ganglion. beta-subunits beta2 and beta4 showed high expression in brain and trigeminal ganglion and some in cerebral vessels while beta1 showed highest expression in blood vessels. beta3 was found only in testis and possibly brain. A novel splice variant X2(+92) was found, which generates a stop codon in the intracellular C-terminal part of the protein. This variant appears non-functional as a homomer but may modulate the function of other splice-variants when expressed in Xenopus oocytes. In conclusion a great number of splice variant and beta-subunit combinations likely exist, being differentially expressed among nervous and vascular tissues.     

成年鼠缺血性脑损伤诱导nestin的表达

应用免疫组化和免疫荧光双标技术结合激光共聚焦扫描显微镜,观察缺血性脑损伤后脑内nestin的表达及其细胞类型。实验观察结果为,再灌后1天,在缺血中心区可见nestin阳性突起;再灌后3天和1周时,除缺血中心区外,周边I、Ⅱ、Ⅲ区均有nestin阳性突起主要与GFAP共存;2周时,nestin阳性突起变粗、变长,并与NSE的共存明显增多。上述研究结果提示,脑缺血可诱导大鼠脑缺血区域表达nestin,该表达可能与神经细胞的修复有关。     

Localization of Angiopoietin-1 and Tie2 Immunoreactivity in Rodent Ependyma and Adjacent Blood Vessels Suggests Functional Relationships

Angiopoietin-1 (Angpt1; previously Ang-1) participates in vascular maintenance and remodeling. In the current study, we investigated the distribution of Angpt1 protein in rat brain. We detected Angpt1 immunoreactivity (IR) in cerebral blood vessels, cuboidal ependyma, and tanycytes, which are specialized hypothalamic bipolar ependymal cells. We also evaluated patterns of IR of endothelium-specific receptor tyrosine kinase 2 (Tie2, the receptor for Angpt1). Tie2 IR was present in Angpt1-immunoreactive cuboidal ependyma in a membranous pattern, suggesting an autocrine or paracrine role for Angpt1–Tie2. Tie2 IR was also associated with peri-ependymal blood vessels, some of which were contacted by tips of Angpt1-immunoreactive tanycyte processes, implying a potential functional ligand−receptor interaction mediating communication between the cerebrospinal fluid and vascular compartments. Because we previously found that cerebral Angpt1 expression was modulated by 17β-estradiol (E2), and because some tanycyte functions are modulated by E2, we tested the hypothesis that E2 affects ependymal and tanycyte Angpt1 expression in vivo. No gross E2 effect on the ependymal pattern of Angpt1 IR or cerebral Angpt1 protein content was observed. (J Histochem Cytochem 58:53–60, 2010)     

Selective ablation of alphav integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death

Mouse embryos genetically null for all alphav integrins develop intracerebral hemorrhage owing to defective interactions between blood vessels and brain parenchymal cells. Here, we have used conditional knockout technology to address whether the cerebral hemorrhage is due to primary defects in vascular or neural cell types. We show that ablating alphav expression in the vascular endothelium has no detectable effect on cerebral blood vessel development, whereas deletion of alphav expression in central nervous system glial cells leads to embryonic and neonatal cerebral hemorrhage. Conditional deletion of alphav integrin in both central nervous system glia and neurons also leads to cerebral hemorrhage, but additionally to severe neurological defects. Approximately 30% of these mutants develop seizures and die by 4 weeks of age. The remaining mutants survive for several months, but develop axonal deterioration in the spinal cord and cerebellum, leading to ataxia and loss of hindlimb coordination. Collectively, these data provide evidence that alphav integrins on embryonic central nervous system neural cells, particularly glia, are necessary for proper cerebral blood vessel development, and also reveal a novel function for alphav integrins expressed on axons in the postnatal central nervous system.     

Post-ischemic hypothermia promotes generation of neural cells and reduces apoptosis by Bcl-2 in the striatum of neonatal rat brain

Hypothermia is a potential therapy for cerebral hypoxic ischemic injury in adults and neonates. However, the mechanism of hypothermia neuroprotection after hypoxic-ischemia (HI) on the developing rat brain remains unclear. In this research, 7-day-old rats were subjected to left carotid artery ligation followed by 8% oxygen for 2h. They were divided into hypothermia (rectal temperature, 32-33°C for 24h) and normothermia (36-37°C for 24h) groups immediately after hypoxia-ischemia. All rats were given 50mg/kg/day 5-bromodeoxyuridine (BrdU) intraperitoneally at 4-6 days and sacrificed at 1 or 2 weeks after HI. There was a significant decrease in infarct volume in the hypothermia group at 7 days after HI compared with that in the normothermia group. The numbers of nestin-labeled cells did not change greatly, but β-tubulin III (Tuj-1) immuno-positive cells increased significantly in the striatum at 1 and 2 weeks after HI in the hypothermia compared to normothermia group. Neurogenesis was assessed by double immunohistochemical/immunofluorescent labeling of BrdU with nestin, Tuj-1 or microtubule-associated protein 2 (Map-2). Newborn neural progenitors (BrdU(+)-nestin(+)) did not change dramatically, but newborn immature (BrdU(+)-Tuj-1(+)) and mature (BrdU(+)-Map-2(+)) neurons increased significantly in the hypothermia compared with normothermia group. Meanwhile, the apoptosis rate of neural precursors, immature and mature neurons, assessed by double labeling of active Casp-3 with nestin/Tuj-1/Map-2, decreased noticeably in the hypothermia compared with normothermia group. We also found that hypothermia significantly increased expression of Bcl-2, which coexisted with nestin/Tuj-1/Map-2. Inhibition of Bcl-2 expression reversed the decreased apoptosis rate of neural precursors and neurons in hypothermia animal striatum of neonatal rat brain. These results suggest that neuroprotection effects of hypothermia on injured developing rat brain may associate with enhanced generation of neuronal cells and Bcl-2-mediated reduction of apoptosis of these cells. These observations are noteworthy regarding clinical hypothermia therapy following cerebral HI injury during the perinatal period.     

Transplantation of the rat pineal organ to the brain: pinealocyte differentiation and innervation

Summary The pineal organ of neonatal rats was transplanted to the frontal part of the cerebral cortex or the cerebral interhemispheric fissure of an isogenic adult rat to determine whether pineal differentiation and pinealopetal innervation are affected by aberrant neuronal influences. Transplants were fixed for immunohistochemistry at 1, 2 and 6 months after transplantation. When treated with an anti-serotonin antibody, cells in transplants from both locations showed intense immunoreactivity and a morphology comparable to intact pinealocytes, indicating that the transplanted pinealocytes had differentiated normally. Tyrosine hydroxylase immunohistochemistry revealed that new catecholamine fibers of central nervous origin extended only into the periphery and not into the core of transplants grafted within the cortex. However, numerous catecholamine fibers were found in transplants placed in the interhemispheric fissure. These fibers were often accompanied by blood vessels, suggesting that they derived from sympathetic ganglia. Serotonin fibers, which are densely distributed in the cerebral cortex, were seldom found to enter transplants from both locations. These observations indicate that pineal cells express their characteristic properties even when transferred to a foreign milieu and that they do not receive novel innervation from the central nerves that normally do not innervate the intact pineal body; the transplant thereby retains the property of selective pinealopetal innervation.     

Immunohistochemical demonstration of Angiopoietin-2 in lymphatic vascular development

The cellular expression of Angiopoietin-2 (Ang2) was studied during lymphatic development in mouse by immunohistochemistry and compared to that of lymphatic endothelial markers. At the earliest stage of lymphvasculogenesis, Prox1-identified lymphatic precursor cells of the cardinal vein displayed an intense immunoreaction for Ang2 in their cytoplasm, implying that Ang2 may adjust lymphatic specification and sprouting from the veins under the control of Prox1. Thereafter, Ang2 was constantly expressed in Prox1 and/or LYVE-1-immunopositive endothelial cells of lymphatic sacs and vessels, ranging from lymphatic capillaries to collectors, throughout embryonic and neonatal development, and the lymphatic endothelial cells simultaneously exhibited immunoreactivity to Tie2, a primary receptor for angiopoietins. These results suggest that lymphatic endothelial cells may regulate lymphatic development via their own Ang2-Tie2 signaling. Ang2 is further immunolocalized in the developing blood vessels including hepatic sinusoids, adrenal medullary vasculature and postnatal pulmonary vessels, thereby indicating that the blood vessels, which undergo vascular remodeling and sudden alteration of blood flow during the development, are also likely to express Ang2. The present study is first to demonstrate Ang2 expression in the lymphatic endothelial cells during development, and consequently Ang2 is regarded as a molecular profile of the developing lymphatic endothelial cells required for lymphatic vascular organization.     

Transient expression of Bis protein in midline radial glia in developing rat brainstem and spinal cord

Bis (Bcl-2 interacting death suppressor) has been reported to contribute to the differentiation and maturation of specific neuronal populations in the developing rat forebrain, in addition to its well-established functions as a stress or survival-related protein. In the present study, we have analyzed the expression of Bis in the rat brainstem and cervical spinal cord during development by using immunohistochemistry. Bis immunoreactivity was detected in radial glial cells flanking the midline from embryonic day 14. During embryonic and early postnatal development, Bis expression persisted in differentiating radial glia at the midline but disappeared first in the spinal cord by postnatal day 7 (P7) and later also in the brainstem by P14. Bis expression was restricted to a subpopulation of the midline radial glia, i.e., the dorsal midline of the midbrain and spinal cord and the ventral midline of the hindbrain, which were double- or triple-labeled with vimentin and nestin, markers for radial glia, and S100B. However, these markers also labeled all radial glia including the ventral midline glia in the midbrain and spinal cord, with Bis being absent from these structures. In addition, the dorsal midline glia in the midbrain and spinal cord expressed Bis prior to the timing of expression for radial glial markers. Therefore, our results demonstrate the early and transient expression of Bis in the subpopulation of midline glia in the developing brainstem and spinal cord, suggesting that Bis has a unique role in association with the radial glial cells in the developing central nervous system. This research was supported by a grant (10029970) from the Ministry of Knowledge Economy, The Republic of Korea and by a grant (M103KV010010-08 K2201-01010) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, The Republic of Korea.     

Reduced subcommissural organ glycoprotein immunoreactivity precedes aqueduct closure and ventricular dilatation in H-Tx rat hydrocephalus

The H-Tx rat has fetal-onset hydrocephalus associated with closure of the cerebral aqueduct and a reduction in the secretory cells of the subcommissural organ (SCO), a circumventricular organ situated in the dorsal wall of the cerebral aqueduct. The objective of this study was to determine the role of the SCO in hydrocephalus pathogenesis. Serial brain sections through aqueduct regions containing the SCO from H-Tx rats, together with non-hydrocephalic Fischer F344 rats, were studied at E16, before hydrocephalus onset, at E17, the beginning of onset, and at P0 when the hydrocephalus was overt. Tissues were immunostained by AFRU, an antibody against the SCO glycoprotein, and for the intermediate filament nestin. The area of SCO cells with AFRU immunostaining and the severity of lateral ventricle dilatation were quantified by image analysis. At E16 all fetuses had distinct SCO ependymal cells, open aqueducts and normal lateral ventricles. The H-Tx fetuses fell into two groups with large areas and small areas of AFRU immunoreactivity, all with a full complement of SCO cells. By E17, fetuses with small areas of immunoreactivity had reduced numbers of tall SCO secretory cells, and most had aqueducts closed posteriorly and dilated ventricles. Three additional fetuses with small areas of immunoreactivity had narrow but patent aqueducts and normal ventricles, and another had an open aqueduct and dilated ventricles. At P0, pups previously identified as hydrocephalic had small areas of AFRU immunoreactivity, an aqueduct that was closed anteriorly but open posteriorly, ventricular dilatation, and an absence of SCO secretory cells. The aqueduct even when closed was lined by typical ependymal cells throughout. Decreased nestin immunostaining accompanied the SCO changes. It is concluded that reduced SCO glycoprotein immunoreactivity precedes both aqueduct closure and expansion of the lateral ventricles in the H-Tx rat.Funding was provided by the National Institutes of Health (NS40359). K.C.S. was supported by the University of Florida Scholars Program and Sigma Xi Grants-in-Aid