Paul A. Knepper

The cause of Chiari II malformation: a unified theory.

The cause of the Chiari II hindbrain deformity in children born with a myelomeningocele can be explained by the lack of distention of the embryonic ventricular system. Defective occlusion and an open neural tube precludes the accumulation of fluid and pressure within the cranial vesicles. This distention is critical to normal brain development. The small posterior fossa, cerebral disorganization, and lückenschädel are the result.

Intraocular pressure and glycosaminoglycan distribution in the rabbit eye: Effect of age and dexamethasone

Abstract The effect of dexamethasone on intraocular pressure was studied in young and old rabbits and was related to the concentration and distribution of the glycosaminoglycans obtained from the proteoglycan in the anterior segment of the eye (central cornea, peripheral cornea, sclera and iris). Topical application of dexamethasone resulted in an increase of intraocular pressure of young rabbits but had no effect on the intraocular pressure of old rabbits. The changes in the relative distribution of the glycosaminoglycans components were observed as a function of age and/or dexamethasone treatment. The ratio of non-uronic acid containing glycosaminoglycans (keratan sulfate) to the uronic acid containing glycosaminoglycans decreased with age and was reversed by dexamethasone treatment of the aged animal. Thus, the distribution of the glycosaminoglycans induced by dexamethasone treatment may modulate intraocular pressure.

Role of the Neural Crest in Anterior Segment Development and Disease

Certain eye and associated systemic developmental anomalies are apparently related by virture of a common neural crest origin. The development of the anterior segment is extremely complex and is dependent upon the presence or absence of certain local factors (including extracellular matrices and glycoproteins), inductors, receptors, and specific time sequencing. Understanding anterior segment anomalies and their systemic associations requires an understanding of neural crest proliferation and migration patterns; and they may be unified under the designation of neurocristopathies. Goldenhars syndrome, not previously considered a neurocristopathy, may be considered one on the basis of the relationship between clinical findings and neural crest embryology.

Myelomeningocele and Waardenburg syndrome (type 3) in patients with interstitial deletions of 2q35 and the PAX3 gene: Possible digenic inheritance of a neural tube defect

From a spina bifida clinic we have identified two patients with a syndrome of myelomeningocele and Waardenburg syndrome type 3 (WS3). The patients each possess a single, de novo, interstitial deletion of chromosome 2 (2q35-36.2), including the PAX3 gene. Deletion of PAX3 was confirmed by fluorescence in situ hybridization (FISH). Analysis with PAX3 and flanking microsatellites shows that the deleted interval of chromosome 2 is of paternal origin and is at least 2 and 6 cM in the two patients. Interstitial deletions in this region result in the Waardenburg syndrome (WS1), but have not been associated with neural tube defects (NTDs). Although other etiologies have not been formally excluded, these patients raise the possibility of a digenic etiology of their NTDs via a genetic interaction of the deleted PAX3 gene with a second unidentified locus.

Aqueous outflow pathway glycosaminoglycans.

Abstract The acidic glycosaminoglycan distribution patterns of the aqueous outflow pathway, the iris-ciliary body, and sclera of the New Zealand Red rabbit were identified by analysis of the glycosaminoglycan moieties and by the use of zone electorphoresis. Alcian blue positive bands on cellulose acetate membranes were characterized by a determination of their electrophoretic mobility in two electrolyte systems and by a comparison to the electrophoretic mobility of standard reference glycosaminoglycans. To verify the identity of each band, specific glycosaminoglycan degrading enzymes were used to remove the glycosaminoglycans. Glycosaminoglycan samples equivalent to 5 μg uronic acid were treated with hyaluronate lyase, testicular hyaluronidase, chondroitin AC lyase and chondroitin ABC lyase. The glycosaminoglycans resistant to chondroitin ABC lyase were treated with nitrous acid and tested for solubility in cetyl pyridinium chloride. The scleral distribution pattern was hyaluronic acid, chondroitin sulfate and hybrid dermatan sulfate-chondroitin sulfate. The aqueous outflow pathway and iris-ciliary body distribution patterns were hyaluronic acid, keratan sulfate, heparan sulfate and hybrid dermatan sulfate-chondroitin sulfate.

Aqueous humor sCD44 concentration and visual field loss in primary open-angle glaucoma

PurposeTo correlate aqueous humor soluble CD44 (sCD44) concentration, visual field loss, and glaucoma risk factors in primary open-angle glaucoma (POAG) patients. MethodsAqueous samples were obtained by paracentesis from normal and glaucoma patients who were undergoing elective surgery and analyzed for sCD44 concentration by enzyme-linked immunosorbent assay. ResultsIn normal aqueous (n=124) the sCD44 concentration was 5.88±0.27 ng/mL, whereas in POAG aqueous (n=90) the sCD44 concentration was 12.76±0.66 ng/mL, a 2.2-fold increase (P<0.000001). In POAG patients with prior successful filtration surgery (n=13), the sCD44 concentration was decreased by 43% to 7.32±1.44 (P=0.001) in comparison with POAG patients without filtration surgery; however, the sCD44 concentration in the prior successful filtration subgroup with no medications and normal intraocular pressure was 12.62±3.81 (P=0.05) compared with normal. The sCD44 concentration of normal pressure glaucoma patients was 9.19±1.75 ng/mL, a 1.6-fold increase compared with normal (P=0.02). Race and intraocular pressure pulse amplitude were significant POAG risk factors in this cohort of patients. In both normal and POAG patients with mild and moderate visual field loss, sCD44 concentration was greater in African Americans than in whites (P=0.04) ConclusionssCD44 concentration in the aqueous of POAG patients correlated with the severity of visual field loss in all stages in white patients and in mild to moderate stages in African American patients. sCD44 concentration in aqueous is a possible protein biomarker of visual field loss in POAG.

Role of Complex Carbohydrates and Neurulation

Extracellular and cell-coat complex carbohydrates play a major role in critical cellular functions, e.g., cell recognition and cell-to-cell adherence, which are involved in neurulation. To date, available evidence suggests that neurulation is a combination of intercellular and extracellular biochemical and morphological events which are under the direction of the genome. To define the carbohydrate components that are essential to neurulation, normal C-57 and abnormal splotch (Sp/Sp) mouse embryos were studied using fluorescein-labeled lectins and computer-aided microspectrophotometric analysis of hyaluronate lyase and chondroitin ABC lyase-sensitive Alcian blue staining. Preliminary results of these studies indicate that the neuroepithelium of the splotch Sp/Sp mutant, a genetic model of a primary neural tube defect, is characterized by alterations in the type and amount of glycoconjugates and in the concentration of individual glycosaminoglycans. This review of mammalian neurulation discusses the importance of complex carbohydrates on the cell surface of the neuroepithelium of the closing neural fold.

Microanalysis of glycosaminoglycans

Abstract A sensitive and versatile method for the qualitative and quantitative determination of glycosaminoglycans (GAGs) is described. An enriched GAG fraction was subjected to nuclease enzyme treatment and to an appropriate sequence of GAG degrading enzymes—Streptomyces hyaluronidase, chondroitinase AC and ABC, and endo-β- d -galactosidase—and nitrous acid treatment. To determine the result of each degradative procedure, the remaining GAG polymers were subjected to cellulose acetate electrophoresis. The combination of sequential degradation and the monitoring of each step by electrophoresis and densitometry permitted the identification and quantitation of all the GAGs on a microscale basis.

Effect of chondroitin sulfate on intraocular pressure in rats.

PURPOSE To study the effect of intracameral injections of chondroitin sulfate (CS) on intraocular pressure (IOP), retinal function, and histology in rats. METHODS Acute or chronic injections of CS were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with vehicle. IOP was daily or weekly assessed by a tonometer. Retinal function was assessed by scotopic electroretinography (ERG) and the visual pathway by flash visual evoked potentials (VEPs), whereas the retinal and optic nerve head structure were examined by histologic analysis. RESULTS A single injection of 8 mg (but not 2 or 4 mg) CS induced a significant increase of IOP. The increase of IOP induced by a single injection of 8 mg CS lasted for 7 days, whereas chronic (weekly) administration during 10 weeks induced a significant and sustained increase in IOP compared with eyes injected with vehicle. A significant decrease of scotopic ERG a- and b- wave amplitude was observed after 6 and 10 weeks of CS administration. Moreover, a significant decrease in scotopic flash VEP N2-P2 component amplitude was observed in eyes treated with CS for 6 and 10 weeks. A significant loss of ganglion cell layer cells and optic nerve axons was observed in eyes receiving CS for 10 weeks. CONCLUSIONS These results suggest that exogenous CS simulates the accumulation of CS in primary open-angle glaucoma and that increased amounts of CS could play a key role in the IOP dysregulation characteristic of glaucoma.

Reconstitution of trabecular meshwork GAGs: Influence of hyaluronic acid and chondroitin sulfate on flow rates

Purpose:This study was undertaken to determine whether the concentration of hyaluronic acid (HA) and of chondroitin sulfate (CS) occurring in the normal and the primary open-angle glaucoma (POAG) trabecular meshwork (TM) influences flow rates in vitro as a function of pressure. Methods:We tested 100, 500, and 4000 kDa molecular weight HA, CS, reconstituted normal and POAG TM HA-CS and juxtacanalicular connective tissue (JCT) HA-CS in a micro test chamber to determine initial and steady-state flow rates. The resistance and permeability (Ko) were calculated; Linear Newtonian mechanics were used to determine the possible contributions of the hydrophobic interactions of HA. Results:Initial flow rates increased in the pressure range of 5 to 20 mm Hg for the three HA preparations and the flow rates declined in the pressure range of 20 to 40 mm Hg. Flow rates of reconstituted normal TM and JCT were optimum at 10 mm Hg and then declined with increasing pressure. Flow rates of reconstituted POAG TM and JCT were optimum only at 5 mm Hg and then declined. The steady-state rate of POAG JCT HA-CS at 10 mm Hg was slow: the transition time (ie, the time required to start an increase in flow rate) was 29 hours and the lag time (ie, the time required to obtain steady-state flow rate) was 17 hours. The maximum flow rate in POAG JCT HA-CS decreased by 37.2% from the normal JCT HA-CS. The calculated resistance of reconstituted POAG JCT HA-CS was approximately 18% of the total resistance of the human JCT compared with 10% in the normal JCT. Conclusions:Hyaluronic acid and CS contribute to flow resistance and influence flow rate in vitro. The influence of HA is particularly sensitive to an increase in the pressure gradient, which may be caused by unfolding of the hydrophobic interactions of HA polymers that further entangles the HA polymer. The POAG JCT HA-CS concentrations represent a significant factor in outflow resistance in POAG, particularly at higher pressures.