Cryoembedding and sectioning of cochleas for immunocytochemistry and in situ hybridization.

Current emphasis on biochemical and molecular aspects of cochlear anatomy underscores the necessity for high quality cryostat sections of the inner ear. The large volume of fluid space within the cochlea makes cryoembedding and sectioning of the organ more problematic than that of other, more homogeneous tissues. Our method for cryoembedding of cochleas for immunocytochemistry and in situ hybridization uses slow infiltration with increasing concentrations of sucrose followed by degassed embedding medium before final orientation and freezing. This method permits high quality cryosections to be cut which preserve overall structure and cellular resolution.

A temporospatial map of adhesive molecules in the organ of Corti of the mouse cochlea.

In the nervous system, several classes of cell-surface and extracellular matrix molecules have been implicated in processes such as neural growth, fasciculation, pathfinding, target recognition and synaptogenesis, which require cell-to-cell or cell-to-substrate binding. In the developing mouse cochlea, little is known about the types of cell-surface and extracellular matrix molecules existing along the neural growth paths or their possible roles in development. Whole mount and sectioned cochlear tissue were immunolabeled for six different adhesive molecules - neural cell adhesion molecule (NCAM), polysialic acid (PSA), neural cell adhesion molecule L1, E-cadherin, syndecan-1 and tenascin-C. A temporospatial map of adhesive molecule distribution in the basal turns of the mouse cochlea was generated. Distributions of adhesive molecules were compared to each other and to the known progress of neural development in the region. This comparison demonstrated differences in the complements of adhesive molecules between the inner and outer hair cell regions, and variations in the expressions of adhesion molecules among different types of nerve fibers. In addition, developmental changes in the adhesive environment around and beneath the outer hair cells coincided with the known timing of the appearance of morphologically defined efferent synapses. These observations raise the possibility that molecular differences at the cell surface of inner and outer hair cells are one way that ingrowing neurites distinguish different environments to determine their growth routes and synaptic partners in the cochlea. In addition these observations demonstrate the potential for differential signaling of afferent and efferent innervation by altering the microenvironments in which synapses are formed.

Cholecystokinin increases bile acid synthesis with total parenteral nutrition but does not prevent stone formation.

Total parenteral nutrition (TPN) is associated with cholestasis and gallstones. Gallbladder stasis may be important in the development of gallstones, and cholecystokinin (CCK) to stimulate gallbladder contraction has been proposed as a treatment to prevent this complication. We studied in vivo bile acid synthesis and bile acid output in miniswine on TPN to test whether daily CCK improves bile acid output and normalizes bile acid profiles with TPN. Nine miniswine were nutritionally maintained with TPN for 4 weeks; four pigs received CCK (0.1 mg/kg) iv daily. In vivo bile acid synthesis was measured with injection of 7 alpha-tritiated cholesterol. An increase in tritiated water reflects the activity of 7 alpha-hydroxylation, the rate-limiting step in bile acid synthesis. At the end of 4 weeks, bile was collected and bile acid output and bile salt profiles were determined. One of five animals on TPN developed gallstones while two of four receiving daily CCK developed stones. In vivo bile acid synthesis decreased with TPN (controls, 63 +/- 9 mg/24 hr versus TPN, 13 +/- 4 mg/24 hr) and increased in TPN animals with CCK treatment (TPN-CCK, 105 +/- 35 mg/24 hr). Bile acid profiles are changed with TPN with more secondary bile acids, this was not improved with CCK. CCK improved bile acid synthesis and bile acid output but failed to prevent gallstone formation or normalize bile salt profiles. In addition to promoting gallbladder contraction, CCK may have a stimulatory effect on bile acid synthesis. CCK alone did not prevent gallstone formation.

Reduced ileal taurocholate absorption with total parenteral nutrition.

Total parenteral nutrition is associated with decreased primary bile acid output. This may result from reduced synthesis or decreased absorption of primary bile acids in the enterohepatic circulation. Ileal atrophy occurs with total parenteral nutrition, and we postulated that absorption of primary bile acids is reduced. Ileal absorption of taurocholate was investigated in miniswine on total parenteral nutrition and this was compared to orally fed animals. Following cholecystectomy and bile duct cannulation, 20 cm, of ileum was perfused with taurocholate. Since miniswine have no significant endogenous taurocholate secretion, the measured taurocholate output reflected ileal absorption. To examine uptake and secretion of taurocholate by the liver, pigs on total parenteral nutrition and controls were infused with intravenous taurocholate (0.5 to 2.5 mumole/kg/min), and bile acid output was determined. Baseline bile acid output was decreased in animals on total parenteral nutrition, yet the response to intravenous taurocholate was similar to controls. When taurocholate was perfused in the ileum, taurocholate output was markedly different. Taurocholate output (pmole/cm ileum/min) at the taurocholate perfusate concentrations of 3 and 20 mM was, respectively, 65 +/- 14 and 183 +/- 63 for controls and 18 +/- 3 and 63 +/- 17 for miniswine on total parenteral nutrition. The latter group's output was significantly lower, at P < 0.05. Both groups of pigs had equal increases in bile acid output with intravenous infusion of taurocholate, suggesting normal hepatic uptake and secretion. Ileal perfusion with taurocholate, however, resulted in decreased taurocholate output with total parenteral nutrition due to decreased ileal absorption of bile acids.(ABSTRACT TRUNCATED AT 250 WORDS)