Hydrocephalus In Rats Research Articles

Evaluation of the behavioral, histopathological, and immunohistochemical effects resulting from ventriculosubcutaneous shunt obstruction in kaolin-induced hydrocephalus in rats.

Hydrocephalus is a brain disease prevalent in the pediatric population that presents complex pathophysiology and multiple etiologies. The best treatmentis still ventricular shunting. Mechanical obstruction is the most frequent complication, but the resulting pathological effects are still unknown. Evaluation and comparison of clinical, histopathological, and immunohistochemical aspects in the acute phase of experimental hydrocephalus induced by kaolin, after treatment with adapted shunt, and after shunt obstruction and posterior disobstruction. Wistar rats aged 7days were used and divided into 4 groups: control group without kaolin injection (n = 6), untreated hydrocephalic group (n = 5), hydrocephalic group treated with ventriculosubcutaneous shunt (DVSC) (n = 7), and hydrocephalic group treated with shunt, posteriorly obstructed and disobstructed (n = 5). The animals were submitted to memory and spatial learning evaluation through the Morris water maze test. The rats were sacrificed at 28days of age and histological analysis of the brains was performed with luxol fast blue, in addition to immunohistochemical analysis in order to evaluate reactive astrocytosis, inflammation, neuronal labeling, and apoptotic activity. The group with shunt obstruction had worse performance in memory tests. Reactive astrocytosis was more evident in this group, as was the inflammatory response. Obstruction of the shunt results in impaired performance of behavioral tests and causes irreversible histopathological changes when compared to findings in the group with treated hydrocephalus, even after unblocking the system. The developed model is feasible and efficient in simulating the clinical context of shunt dysfunction.

Iron-Induced Hydrocephalus: the Role of Choroid Plexus Stromal Macrophages.

Evidence indicates that erythrocyte-derived iron and inflammation both play a role in intraventricular hemorrhage-induced brain injury including hydrocephalus. Many immune-associated cells, primarily stromal macrophages, reside at the choroid plexus where they are involved in inflammatory responses and antigen presentation. However, whether intraventricular iron impacts those stromal cells is unknown. The aim of this study was to evaluate the relationship between choroid plexus stromal macrophages and iron-induced hydrocephalus in rats and the impact of minocycline and clodronate liposomes on those changes. Aged (18-month-old) and young (3-month-old) male Fischer 344 rats were used to study choroid plexus stromal macrophages. Rats underwent intraventricular iron injection to induce hydrocephalus and treated with either minocycline, a microglia/macrophage inhibitor, or clodronate liposomes, a macrophage depleting agent. Ventricular volume was measured using magnetic resonance imaging, and stromal macrophages were quantified by immunofluorescence staining. We found that stromal macrophages accounted for about 10% of the total choroid plexus cells with more in aged rats. In both aged and young rats, intraventricular iron injection resulted in hydrocephalus and increased stromal macrophage number. Minocycline or clodronate liposomes ameliorated iron-induced hydrocephalus and the increase in stromal macrophages. In conclusion, stromal macrophages account for ~10% of all choroid plexus cells, with more in aged rats. Treatments targeting macrophages (minocycline and clodronate liposomes) are associated with reduced iron-induced hydrocephalus.

UPA alleviates kaolin-induced hydrocephalus by promoting the release and activation of hepatocyte growth factor in rats

Urokinase-type plasminogen activator (uPA) was demonstrated to alleviate kaolin-induced communicating hydrocephalus via inhibiting subarachnoid space fibrosis, but the exact mechanism remains elusive. Thus, this study was designed to investigate if hepatocyte growth factor (HGF), which plays a vital role in uPA-triggered inhibiting of fibrosis in multiple systems, is involved in this process in hydrocephalus. There were 2 parts in this study. First, hydrocephalus was induced in rats by basal cistern injection of kaolin. Then rats were treated with saline or uPA and brain tissue and CSF were collected for Western blot and enzyme-linked immuno sorbent assay (ELISA) four days later. Second, kaolin-induced hydrocephalus rats were treated with saline, uPA, uPA + PHA665752 (antagonist of HGF) or PHA665752. Some animals received MRI four weeks later and brains were used for immunofluorescence. The others were euthanized four days later for ELISA. Both levels of total and activated HGF in the CSF was increased after uPA injections, but related mRNA expression of HGF showed no statistical significance when compared with the control group. Further, the effects of uPA that alleviating ventricular enlargement, subarachnoid fibrosis and reactive astrocytosis were partially reversed by PHA665752. Moreover, PHA665752 partially abolished uPA-induced reduction of transforming growth factor- β1(TGF- β1) level in CSF. Our data suggest that uPA effectively inhibited subarachnoid fibrosis and restricted the development of communicating hydrocephalus in rats in part by promoting HGF release and activation, which may further regulate the TGF-β1 expression in CSF.

Evaluation of the effects of quercetin on brain lesions secondary to experimental hydrocephalus in rats

Hydrocephaly is a disease that affects not only the dynamics of the cerebrospinal fluid, but also other structures of the central nervous system. Although shunt is effective in reducing ventriculomegaly, many neurological damages are not reversed with surgery. Several studies demonstrate that oxidative stress is involved in the genesis of hydrocephalus lesions. Evaluate the neuroprotective response of quercetin in hydrocephalus. Male newborns rats were used, which received the 15% kaolin injection in the cisterna magna for induction of hydrocephalus. They were divided into control group (C), untreated hydrocephalic (HN), shunted hydrocephalic (HD), hydrocephalic treated with distilled water (HA), hydrocephalic treated with distilled water and shunt (HDA), hydrocephalic treated with quercetin peritoneal (HQp), hydrocephalic treated with quercetin peritoneal and shunt (HDQp), hydrocephalic treated with quercetin by gavage (HQg), and hydrocephalus treated with quercetin by gavage and shunt (HDQg). Quercetin significantly improved the immunohistochemical markers, mainly caspase and GFAP. There were no significant changes in clinical/behavioral assessment. The use of isolated quercetin does not alter the volume and ventricular size, and the realization of ventriculo-subcutaneous shunt in newborn rats with hydrocephalus presents a high morbi-mortality. The use of quercetin shows laboratory improvement of the effects of glial lesion and corpus callosum fibers and is therefore not justified by the use of the routine substance as neuroprotective.

Intraventricular administration of urokinase as a novel therapeutic approach for communicating hydrocephalus

Fibrosis of the subarachnoid space (SAS) after infection, inflammation, or hemorrhage can impair cerebrospinal fluid absorption and circulation, causing diffuse ventricular dilatation. In the present study, we tested the hypothesis that urokinase (also known as urokinase-type plasminogen activator [uPA]), a fibrinolytic agent, attenuates fibrosis and ventriculomegaly in a rat model of kaolin-induced communicating hydrocephalus and thus may have potential as a therapy for these conditions. Thirty microliters of sterile 25% kaolin suspension was injected into the basal cisterns of adult Sprague-Dawley rats to induce hydrocephalus, and 2 intraventricular injections of either uPA or vehicle (saline) were administered immediately and 3 days thereafter. Ventricular volumes were measured by magnetic resonance imaging (MRI) on days 3, 14, and 28 after kaolin injection. Fibrosis and reactive astrogliosis were evaluated on day 28 by immunofluorescence and Western blotting. Neurocognitive features were tested using the Morris water maze from days 23 to 28. MRI analysis demonstrated that kaolin administration successfully induced hydrocephalus in rats and that uPA treatment significantly attenuated ventricular enlargement. In addition, uPA inhibited the deposition of laminin and fibronectin, extracellular matrix molecules, in the SAS, attenuated gliosis, and improved learning and memory in kaolin-treated rats. Therefore, we concluded that uPA prevents the development of kaolin-induced communicating hydrocephalus by preventing the development of subarachnoid fibrosis and by eliciting improvements in neurocognition. The results of this study indicate that uPA may be a novel clinical therapy for communicating hydrocephalus.

An experiment model of obstructive hydrocephalus in the rats: injecting the astrocytes into the lateral ventricle

Objective To establish the animal model of obstructive hydrocephalus in rat by injecting lateral ventricle astrocytes from rat. Methods Three different concentrations of 20 μl astrocytes suspension with 5×107, 5×108, 5×109 cells per milliliter were injected into the right lateral ventricle of group A, B, C through stereotactic puncture, respectively, while 20 μl sterile phosphate buffer saline (PBS) solution was injected into the right lateral ventricle of control group. The magnetie resonance imaging (MRI) was performed to obtain T2 weighted brain images at 7, 14, 21, 28, 35 days after injection. Measure the maximum transverse diameter of lateral ventricle through coronary position images to determine the degree of hydrocephalus. Results The mean value of maximum transverse diameter in the control group, group A, group B and group C was (0.11±0.01), (1.01±0.78), (1.83±0.23), (1.89±0.21) mm at 35th day after injection, respectively. Compared with group A, group B and group C, the maximum transverse diameter of control group was statistically different(P 0.05). Conclusion The animal model of obstructive hydrocephalus in rat can be successfully established by injecting lateral ventricle astrocytes. Key words: Hydrocephalus; Rats; Models, animal; Astrocytes

Acetazolamide Attenuates Thrombin-Induced Hydrocephalus.

Our previous studies demonstrated that thrombin is an important factor in brain injury after intracerebral and intraventricular hemorrhage. This study examined the effect of acetazolamide, a carbonic anhydrase inhibitor, on thrombin-induced hydrocephalus. There were two parts in this study. First, rats had an injection of either 50 μl saline or 3 U thrombin into the right lateral ventricle. Second, rats had an injection of 3 U thrombin into the right lateral ventricle and were treated with either vehicle or acetazolamide (30 mg/kg, intraperitoneally (IP)) at 1 h after thrombin infusion. Lateral ventricle volumes were measured in magnetic resonance imaging T2 images and the brains were used for histology analysis at 24 h later. Intraventricular injection of thrombin induced significantly larger ventricle volume (27.8 ± 3.7 vs 8.5 ± 1.3 mm(3), n = 6, p < 0.01) and more ventricular wall damage (the breakdown of the ependymal layer, 20.2 ± 3.1 vs 2.4 ± 0.8 %, n = 6, p < 0.01) compared with saline injection. Acetazolamide treatment (30 mg/kg, IP) markedly attenuated thrombin-induced hydrocephalus (16.1 ± 4.2 mm(3) vs 29.5 ± 5.3 mm(3), n = 6, p < 0.01). These results suggest decreasing CSF production by acetazolamide attenuated thrombin-induced hydrocephalus in rats.

Effects of Aerobic Capacity on Thrombin-Induced Hydrocephalus and White Matter Injury.

We have previously shown that intracerebral hemorrhage-induced brain injury is less in rats bred for high aerobic capacity (high capacity runners; HCR) compared with those bred for low aerobic capacity (low capacity runners; LCRs). Thrombin, an essential component in the coagulation cascade, is produced after cerebral hemorrhage. Intraventricular injection of thrombin causes significant hydrocephalus and white matter damage. In the present study, we examined the effect of exercise capacity on thrombin-induced hydrocephalus and white matter damage. Mid-aged (13-month-old) female LCRs (n = 13) and HCRs (n = 12) rats were used in this study. Rats received an intraventricular injection of thrombin (3 U, 50 μl). All rats underwent magnetic resonance imaging (MRI) at 24 h and were then euthanized for brain histology and Western blot. The mortalities were 20 % in LCRs and 33 % in HCRs after thrombin injection (p > 0.05). No rats died after saline injection. Intraventricular thrombin injection resulted in hydrocephalus and periventricular white matter damage as determined on MRI. In LCR rats, thrombin induced significant ventricle enlargement (23.0 ± 2.3 vs12.8 ± 1.9 mm(3) in LCR saline group; p < 0.01) and white matter lesion (9.3 ± 7.6 vs 0.6 ± 0.5 mm(3) in LCR saline group, p < 0.05). In comparison, in HCR rats thrombin induced less ventricular enlargement (17.3 ± 3.9 vs 23.0 ± 2.3 mm(3) in LCRs, p < 0.01) and smaller white matter lesions (2.6 ± 1.2 mm(3) vs 9.3 ± 7.6 mm(3) in LCRs, p < 0.05). In LCR rats, there was also upregulation of heat shock protein-32, a stress marker, and microglial activation in the periventricular white matter. These changes were significantly reduced in HCR rats. Intraventricular injection of thrombin caused more white matter damage and hydrocephalus in rats with low aerobic capacity. A differential effect of thrombin may contribute to differences in the effects of cerebral hemorrhage with aerobic capacity.

Subarachnoid Hemorrhage-Induced Hydrocephalus in Rats

Hydrocephalus is an important complication of subarachnoid hemorrhage (SAH). We investigated the occurrence of acute hydrocephalus in a rat SAH model. SAH was induced by endovascular perforation in adult male Sprague-Dawley rats (n=36). Sham rats (n=8) underwent the same procedure without perforation. MRI was performed 24 hours after SAH and the volume of the ventricular system and extent of T2* hypointensity lesions were measured. We defined hydrocephalus as ventricular volume > +3 SDs above the mean in sham animals. SAH grade was determined and brains were used for histology, immunohistochemistry, Perls staining, and Western blot analysis. Ventricular wall damage was defined as percentage of ependymal surface disruption. All surviving rats (n=27) after SAH had ventricular enlargement (33.6 ± 4.7 versus 13.5 ± 1.4 mm(3) in sham animals, P<0.01). Ventricular volume correlated with SAH severity (r=0.48; P<0.05). Out of 27 SAH rats, 12 demonstrated hydrocephalus and all had intraventricular blood accumulation. Rats with hydrocephalus had more severe ventricular wall damage (7.4 ± 1.2%) than the sham animals (0.6 ± 0.2%; P<0.01) and rats without hydrocephalus (1.1 ± 0.2%; P<0.01). Periventricular iron deposition was observed and heme oxygenase-1 and Iba-1 expression were markedly increased in hydrocephalus rats. SAH causes ventricular enlargement in a rat endovascular perforation model, with hydrocephalus occurring in 44% of animals at 24 hours. Rats with hydrocephalus had more severe SAH, intraventricular hemorrhage, and greater ventricular wall damage.

0.1T magnetic resonance image in the study of experimental hydrocephalus in rats. Accuracy of the method in the measurements of the ventricular size

To investigate the accuracy of 1.0T Magnetic Resonance Imaging (MRI) to measure the ventricular size in experimental hydrocephalus in pup rats. Wistar rats were subjected to hydrocephalus by intracisternal injection of 20% kaolin (n=13). Ten rats remained uninjected to be used as controls. At the endpoint of experiment animals were submitted to MRI of brain and killed. The ventricular size was assessed using three measures: ventricular ratio (VR), the cortical thickness (Cx) and the ventricles area (VA), performed on photographs of anatomical sections and MRI. The images obtained through MR present enough quality to show the lateral ventricular cavities but not to demonstrate the difference between the cortex and the white matter, as well as the details of the deep structures of the brain. There were no statistically differences between the measures on anatomical sections and MRI of VR and Cx (p=0.9946 and p=0.5992, respectively). There was difference between VA measured on anatomical sections and MRI (p<0.0001). The parameters obtained through 1.0T MRI were sufficient in quality to individualize the ventricular cavities and the cerebral cortex, and to calculate the ventricular ratio in hydrocephalus rats when compared to their respective anatomic slice.

Elevated CSF outflow resistance associated with impaired lymphatic CSF absorption in a rat model of kaolin-induced communicating hydrocephalus.

BackgroundWe recently reported a lymphatic cerebrospinal fluid (CSF) absorption deficit in a kaolin model of communicating hydrocephalus in rats with ventricular expansion correlating negatively with the magnitude of the impediment to lymphatic function. However, it is possible that CSF drainage was not significantly altered if absorption at other sites compensated for the lymphatic defect. The purpose of this study was to investigate the impact of the lymphatic absorption deficit on global CSF absorption (CSF outflow resistance).MethodsKaolin was injected into the basal cisterns of Sprague Dawley rats. The development of hydrocephalus was assessed using magnetic resonance imaging (MRI). In one group of animals at about 3 weeks after injection, the movement of intraventricularly injected iodinated human serum albumin (125I-HSA) into the olfactory turbinates provided an estimate of CSF transport through the cribriform plate into nasal lymphatics (n = 18). Control animals received saline in place of kaolin (n = 10). In a second group at about 3.5 weeks after kaolin injection, intraventricular pressure was measured continuously during infusion of saline into the spinal subarachnoid space at various flow rates (n = 9). CSF outflow resistance was calculated as the slope of the steady-state pressure versus flow rate. Control animals for this group either received no injections (intact: n = 11) or received saline in place of kaolin (n = 8).ResultsCompared to saline injected controls, lateral ventricular volume in the kaolin group was significantly greater (0.087 ± 0.013 ml, n = 27 versus 0.015 ± 0.001 ml, n = 17) and lymphatic function was significantly less (2.14 ± 0.72% injected/g, n = 18 versus 6.38 ± 0.60% injected/g, n = 10). Additionally, the CSF outflow resistance was significantly greater in the kaolin group (0.46 ± 0.04 cm H2O.μL-1.min, n = 9) than in saline injected (0.28 ± 0.03 cm H2O.μL-1.min, n = 8) or intact animals (0.18 ± 0.03 cm H2O.μL-1.min, n = 11). There was a significant positive correlation between CSF outflow resistance and ventricular volume.ConclusionsThe data suggest that the impediment to lymphatic CSF absorption in a kaolin-induced model of communicating hydrocephalus has a significant impact on global CSF absorption. A lymphatic CSF absorption deficit would appear to play some role (either direct or indirect) in the pathogenesis of ventriculomegaly.

Gray matter metabolism in acute and chronic hydrocephalus

Although hydrocephalus is usually considered a disorder of periventricular white matter, disturbance of gray matter is probably also involved. However, so far gray matter metabolism has not been studied in experimental hydrocephalus using high resolution in vivo magnetic resonance spectroscopy (MRS). Therefore 15 rats were made hydrocephalic by injection of 0.1 ml kaolin into the cisterna magna, whereas 10 sham-operated rats served as controls. 1H MRS and magnetic resonance imaging were performed longitudinally in acute hydrocephalus 2 and 4 weeks after kaolin treatment and in chronic hydrocephalus after 6 weeks. Volumes of interest included the gray matter regions cortex, thalamus and hippocampus. In hydrocephalic animals, 1H MRS revealed decreased glutamate levels in all examined areas at all time points. Moreover, in acute hydrocephalus disturbances were noted in the hippocampus with decreased concentrations of N-acetyl aspartate, creatine, inositol and taurine, and in the cortex with decreased taurine levels. A clear lactate peak was detected in CSF spectra from hydrocephalic rats. In addition, T2-weighted images showed increase of free water in the hippocampus. It can be concluded that glutamate metabolism is deranged in gray matter in acute and chronic hydrocephalus in rats. If confirmed in humans, early detection of glutamatergic disturbances and lactate accumulation using in vivo 1H MRS might serve as an indication for surgical treatment of hydrocephalus before irreversible neuronal damage develops.

Evidence for reduced lymphatic CSF absorption in the H-Tx rat hydrocephalus model

BackgroundThere is mounting evidence that spinal fluid absorption takes place not only at the arachnoid villi, but also at several extracranial sites, which might serve as a reserve mechanism for, or be primarily involved in the absorption of CSF in hydrocephalus.MethodsWe compared the nasal lymphatic pathway in congenital Hydrocephalus-Texas (H-Tx) rats in unaffected and affected hydrocephalic (HC) siblings with that of control Sprague Dawley (SD) rat pups. The animals were examined after immediate post mortem injection of Evan's blue dye into the cisterna magna at 6 and 10 days of age. The specimens were evaluated for amount of dye penetration into the nasal passages.ResultsWe found more dye visualization in the olfactory regions of control SD (14/16 at P6, 14/16 at P10) and unaffected H-Tx (13/17 at P6, 13/16 at P10) compared with HC animals (0/14 at P6, 3/15 at P10). This difference was more pronounced at 10 days of age. The dye was not visualized in the cervical lymph nodes or venous channels in these acute experiments.ConclusionThe results of this study suggest that nasal lymphatic cerebrospinal fluid absorption is reduced in the H-Tx rat hydrocephalus model.

The effect of N-methyl- d-aspartate receptor antagonist (memantine) on esophageal and gastric smooth muscle: functional investigation in a rat hydrocephalus model

Object The purpose of this study was to investigate the effect of N-methyl- d-aspartate (NMDA) receptor antagonist on esophageal and gastric smooth muscle reactivity in a rat hydrocephalus model. Material and Methods Hydrocephalus was induced in rats by injection of kaolin into the cisterna magna. Two weeks after the procedure, memantine (20 mg/kg per day, 2 weeks) was given to rats with hydrocephalus in the memantine group (MG). The rest of the rats with hydrocephalus received serum physiologic (hydrocephalus group, HG). The control group (nonhydrocephalic rats, CG) was sham operated. The fourth group consisted of nonhydrocephalic rats with treated memantine (memantine control group, MC). Contractile (KCl, carbachol) and relaxant (isoprenaline, papaverine) esophageal and gastric smooth muscle reactivity were determined by in vitro muscle technique. Results No significant difference was found in the KCl (nonreceptor-mediated)-induced esophageal smooth muscle reactivity among the groups. Carbachol (receptor-mediated)-induced smooth muscle reactivity significantly decreased in HG compared to other groups. The isoprenaline (receptor-mediated)-induced smooth muscle reactivity significantly decreased in HG compared to other groups. No significant difference was found in smooth muscle reactivity to papaverine (nonreceptor-mediated) among the groups. Gastric smooth muscle reactivity to KCl significantly increased in HG compared to other groups. Also, KCl-induced smooth muscle reactivity significantly increased in MG compared to CG and MC. Carbachol-induced smooth muscle reactivity significantly decreased in HG compared to MG, CG, and MC. No significant difference was observed in isoprenaline- and papaverine-induced smooth muscle reactivity among the groups. Conclusion Our findings suggest that memantine may influence esophageal and gastric smooth muscle reactivity in hydrocephalus.

Protein and synthetic polymer injection for induction of obstructive hydrocephalus in rats

BackgroundThe objective of this study was to develop a simple and inexpensive animal model of induced obstructive hydrocephalus with minimal tissue inflammation, as an alternative to kaolin injection.MaterialsTwo-hundred and two male Sprague-Dawley rats aged 3 weeks received intracisternal injections of kaolin (25% suspension), Matrigel, type 1 collagen from rat tail, fibrin glue (Tisseel), n-butyl-cyanoacrylate (NBCA), or ethylene vinyl alcohol copolymer (Onyx-18 and Onyx-34). Magnetic resonance imaging was used to assess ventricle size. Animals were euthanized at 2, 5, 10 and 14 days post-injection for histological analysis.ResultsKaolin was associated with 10% mortality and successful induction of hydrocephalus in 97% of survivors (ventricle area proportion 0.168 ± 0.018). Rapidly hardening agents (fibrin glue, NBCA, vinyl polymer) had high mortality rates and low success rates in survivors. Only Matrigel had relatively low mortality (17%) and moderate success rate (20%). An inflammatory response with macrophages and some lymphocytes was associated with kaolin. There was negligible inflammation associated with Matrigel. A severe inflammatory response with giant cell formation was associated with ethylene vinyl alcohol copolymer.ConclusionKaolin predictably produces moderate to severe hydrocephalus with a mild chronic inflammatory reaction and fibrosis of the leptomeninges. Other synthetic polymers and biopolymers tested are unreliable and cause different types of inflammation.

The secretory ependymal cells of the subcommissural organ: Which role in hydrocephalus?

Ependyma in the central nervous system gives rise to several specialized cell types, including the secretory ependymal cells located in the subcommissural organ. These elongated cells show large cisternae in their cytoplasm, which are filled with material secreted into the cerebrospinal fluid and toward the leptomeningeal spaces. A specific secretion of the subcommissural organ was named SCO-spondin, regarding its marked homology with developmental proteins of the thrombospondin superfamily (presence of thrombospondin type 1 repeats). The ependymal cells of the subcommissural organ and SCO-spondin secretion are suspected to play a crucial role in cerebrospinal fluid flow and/or homeostasis. There is a close correlation between absence of the subcommissural organ and hydrocephalus in rat and mouse strains exhibiting congenital hydrocephalus, and in a number of mice transgenic for developmental genes. The ependymal cells of the subcommissural organ are under research as a key factor in several developmental processes of the central nervous system.

White matter injury in kaolin-induced neonatal rat hydrocephalus

Hydrocephalus is a common neurological condition characterized by obstruction of cerebrospinal fluid (CSF) flow leading to enlargement of CSF-containing ventricular cavities in the brain. The primary target of injury is axons in the periventricular white matter. The stretching of axons leads to dysfunction and ultimately axotomy. Many experiments in mature animals systems indicate that white matter blood flow is decreased. A decrease in oxygen saturation should lead to production of oxygen free radicals and activation of the transcription factor hypoxia inducible factor 1 alpha (HIF). HIF further activates a cascade of cellular responses, notably, vascular endothelial growth factor (VEGF). The main effect of VEGF is to cause production of new blood vessels. Its expression can have an influence on vascular permeability and could play a role in water dysregulation in the hydrocephalic brain. Our first goal was to characterize the structural changes of a neonatal model of kaolin-induced hydrocephalus in rat. Our second goal was to investigate the molecular sequence of events resulting from ischemia. Sprague-Dawley rats underwent kaolin injection into the cisterna magna at postnatal day 1. They had enlarged ventricles by 7 days and severe dilatation by 21 days as assessed by magnetic resonance imaging (MRI) and histology. Periventricular white matter, including corpus callosum and internal capsule, was edematous at 7 days and severely atrophic at 21 days. ELISA and Western blots revealed decreased expression of myelin associated proteins including myelin basic protein (MBP). Pimonidazole hydrochloride (2-nitroimidazole) specifically binds to thiol groups of proteins, peptides and amino acids in a hypoxic environment. Following subcutaneous administration, immunohistochemistry demonstrated hypoxia-associated adducts in periventricular glial cells. Nitrotyrosine, an oxidative stress marker, could be detected by immunohistochemistry extensively in white matter and around some blood vessels. Weak labeling was also seen around large neurons in cerebral cortex. HIF and 4-Hydroxy-2-nonenal (HNE) for endogenous lipid peroxidation are currently under investigation. VEGF immunohistochemistry in normal rats revealed positive cortical neurons in 7-day old rats; this diminished at 21 days. The pattern of expression shifted to white matter glial cells in hydrocephalic rats. VEGF expression determined by ELISA supports those findings. In this model of neonatal kaolin induced hydrocephalus, hypoxia in white matter might contribute to the destructive changes.

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.

Altered expression of sialic acid-bearing glycoconjugates as revealed by lectin binding to choroid plexus in perinatal hydrocephalic rats

Glycoconjugates perform key roles in the development and maintenance of the CNS. A chief saccharide element in brain development is sialic acid, usually occurring as Nacetyl-neuraminic acid. Previous studies by H. Jones et al. demonstrated that glyco-conjugate secretions into CSF, by circumventricular organs like the SCO, may be associated with hydrocephalus development. Therefore, we used 4 lectins (LPA, SNA, MAL-II and WGA), specific for several forms of sialic acid, to probe for alterations in sialic acidbearing glycoconjugate profiles in the CSF-secreting choroid plexus (CP) of the HTx rat hydrocephalus model. Lectins are proteins that bind to carbohydrate residues in an antibody-like manner. We used 3 cohorts of rats: prenatal (20 days of gestation); neonatal (1 day after birth); and postnatal (4 days). Five choroid epithelial domains/ parameters were analyzed: apical cell surface; lateral surface; basal surface; cytoplasm; and all domains considered collectively. Staining intensity was judged visually on a 1+ to 4+ scale, at 400×. Means of sialic acid staining intensity were calculated using 5 animals from each cohort.

Motor evoked potentials in rats with congenital hydrocephalus

Motor evoked potential (MEP) by focal transcranial magnetic stimulation was used to test the functional integrity of the motor cortex in congenital hydrocephalic rats. Magnetic MEPs, using a figure-eight coil above the head, were recorded in the tibialis anterior muscle. The latency of transcranial magnetic MEP was 3.4 msec in nonhydrocephalic rats. In the hydrocephalic rats, the MEP had a lower threshold than in nonhydrocephalic rats, and showed two peaks. Latencies of early and late peaks were 3.9 msec and from 5.4 msec to 10.0 msec, respectively. Our findings suggest that hydrocephalus in rats is associated with changes in pyramidal cell excitability in the motor cortical area, probably induced by the fluctuations in cortical excitability and synaptic interaction in hydrocephalic rats.