MUC1 and Polarity Markers INADL and SCRIB Identify Salivary Ductal Cells.

Current treatments for xerostomia/dry mouth are palliative and largely ineffective. A permanent clinical resolution is being developed to correct hyposalivation using implanted hydrogel-encapsulated salivary human stem/progenitor cells (hS/PCs) to restore functional salivary components and increase salivary flow. Pluripotent epithelial cell populations derived from hS/PCs, representing a basal stem cell population in tissue, can differentiate along either secretory acinar or fluid-transporting ductal lineages. To develop tissue-engineered salivary gland replacement tissues, it is critical to reliably identify cells in tissue and as they enter these alternative lineages. The secreted protein α-amylase, the transcription factor MIST1, and aquaporin-5 are typical markers for acinar cells, and K19 is the classical ductal marker in salivary tissue. We found that early ductal progenitors derived from hS/PCs do not express K19, and thus earlier markers were needed to distinguish these cells from acinar progenitors. Salivary ductal cells express distinct polarity complex proteins that we hypothesized could serve as lineage biomarkers to distinguish ductal cells from acinar cells in differentiating hS/PC populations. Based on our studies of primary salivary tissue, both parotid and submandibular glands, and differentiating hS/PCs, we conclude that the apical marker MUC1 along with the polarity markers INADL/PATJ and SCRIB reliably can identify ductal cells in salivary glands and in ductal progenitor populations of hS/PCs being used for salivary tissue engineering. Other markers of epithelial maturation, including E-cadherin, ZO-1, and partition complex component PAR3, are present in both ductal and acinar cells, where they can serve as general markers of differentiation but not lineage markers.

An improved in vivo method for atrioventricular node ablation via thoracotomy

The atrioventricular (AV) node is permanently damaged in approximately 3 percent of congenital heart surgery operations, requiring implantation of a permanent pacemaker. Improvements in pacemaker design and in alternative treatment modalities require an effective in vivo model of complete heart block (CHB) before testing can be performed in humans. Such a model should enable accurate, reliable, and detectable induction of the surgical pathology. Through our laboratory’s efforts in developing a tissue engineering therapy for CHB, we describe here an improved in vivo model for inducing chronic AV block. The method employs a right thoracotomy in the adult rabbit, from which the right atrial appendage may be retracted to expose an access channel for the AV node. A novel injection device was designed, which both physically restricts needle depth and provides electrical information via electrocardiogram interface. This combination of features provides real-time guidance to the researcher for confirming contact with the AV node, and documents its ablation upon formalin injection. While all animals tested could be induced to acute AV block, those with ECG guidance were more likely to maintain chronic heart block >12 h. Our model enables the researcher to reproduce both CHB and the associated peripheral fibrosis that would be present in an open congenital heart surgery, and which would inevitably impact the design and utility of a tissue engineered AV node replacement.

An improved in vivo method for atrioventricular node ablation via thoracotomy.

The atrioventricular (AV) node is permanently damaged in approximately 3% of congenital heart surgery operations, requiring implantation of a permanent pacemaker. Improvements in pacemaker design and in alternative treatment modalities require an effective in vivo model of complete heart block (CHB) before testing can be performed in humans. Such a model should enable accurate, reliable, and detectable induction of the surgical pathology. Through our laboratory's efforts in developing a tissue engineering therapy for CHB, we describe here an improved in vivo model for inducing chronic AV block. The method employs a right thoracotomy in the adult rabbit, from which the right atrial appendage may be retracted to expose an access channel for the AV node. A novel injection device was designed, which both physically restricts needle depth and provides electrical information via electrocardiogram interface. This combination of features provides real-time guidance to the researcher for confirming contact with the AV node, and documents its ablation upon formalin injection. While all animals tested could be induced to acute AV block, those with ECG guidance were more likely to maintain chronic heart block >12 h. Our model enables the researcher to reproduce both CHB and the associated peripheral fibrosis that would be present in an open congenital heart surgery, and which would inevitably impact the design and utility of a tissue engineered AV node replacement.

Cell-type specific organization of glycine receptor clusters in the mammalian spinal cord.

Glycinergic synapses play a major role in shaping the activity of spinal cord neurons. The spatial organization of postsynaptic receptors is likely to determine many functional parameters at these synapses and is probably related to the integrative capabilities of different neurons. In the present study, we have investigated the organization of gephyrin expression along the dendritic membranes of alpha- and gamma-motoneurons, Ia inhibitory interneurons, and Renshaw cells. Gephyrin is a protein responsible for the postsynaptic clustering of glycine receptors, and the features of gephyrin and glycine receptor alpha(1)-subunit immunofluorescent clusters displayed similar characteristics on ventral horn spinal neurons. However, the density of clusters and their topographical organization and architecture varied widely in different neurons and in different dendritic regions. For motoneurons and Ia inhibitory interneurons, cluster size and complexity increased with distance from the soma, perhaps as a mechanism to enhance the influence of distal synapses. Renshaw cells were special in that they displayed an abundant complement of large and morphologically complex clusters concentrated in their somas and proximal dendrites. Serial electron microscopy confirmed that the various immunoreactivity patterns observed with immunofluorescence accurately parallel the variable organization of pre- and postsynaptic active zones of glycinergic synapses. Finally, synaptic boutons from single-labeled axons of glycinergic neurons (Ia inhibitory interneurons) were also associated with postsynaptic receptor clusters of variable shapes and configurations. Our results indicate that mechanisms regulating receptor clustering do so primarily in the context of the postsynaptic neuron identity and localization in the dendritic arbor.

Experimental seizures in the frog (Rana pipiens).

We investigated the effects of chemical convulsants in the leopard frog. Systemic kainic acid (5-20 mg/kg) caused limbic-like seizures, with staring, catatonia, fasciculations, and severe motor seizures, which were almost always lethal. Intracerebral electroencephalographic (EEG) recordings showed spike or spike-and-wave patterns at 6-8 Hz that decreased in frequency and increased in amplitude, maximal at an electrode in the midline olfactory/telencephalic (OLF-M) region. With time, an interictal pattern of 100-200 microV periodic spikes developed, followed by diffuse suppression of all brain activity. Seizures induced by pentylenetetrazole (150-450 mg/kg) and bicuculline (5-10 mg/kg) were characterized by the abrupt onset of motor activity, which continued intermittently for several hours, followed by recovery. EEG recordings in animals treated with pentylenetetrazole showed rhythmic spike-and-wave bursts at 1.5-3 Hz that were maximal at OLF-M. Recordings from frogs treated with bicuculline showed repetitive 3-6 Hz spike-and-wave discharges maximal at OLF-M that were nearly constant in amplitude and at times became continuous. Strychnine (1-5 mg/kg) caused reversible seizures characterized by tonic extensions of the extremities, that seemed to originate in the spinal cord. Frogs with recurrent seizures from systemic cis-diamminedichloroplatinum II showed 4-8 Hz rhythmic spike-and-wave activity that gradually slowed in frequency and increased in amplitude. Thus, the frog's reactivity to convulsive agents is similar to that of mammals.

Neurotoxic effects of platinum compounds in cultured dorsal root ganglion cells.

The neurotoxic cancer chemotherapeutic agent cis-diamminedichloroplatinum (II) (cis-platin) was tested in a model system of cultured embryonic chick dorsal root ganglion cells, in order to investigate cellular mechanisms of toxicity. At 7.5 ug/ml, the drug caused mild toxicity. At doses of 75 ug/ml, cis-platin was toxic to cultures in 6 hours, in both neuronal and non-neuronal cell populations. After 24 hours of incubation with 75 ug/ml cis-platin, there was extensive cell death. The trans isomer of the drug, trans-platin, was less toxic than cis-platin at similar doses, causing less severe damage to the cells as well as less cell death. With both drugs, abnormalities in patterns of nuclear staining were prominent, whereas neuronal cell membrane staining patterns were less affected. Both drugs seemed to affect non-neuronal cells to a greater extent than neurons. Ultrastructural findings with both drugs included nucleolar segregation; mitochondrial changes were nonspecific. In this in vitro system, both cis- and trans-platin are toxic. The toxicity appears to predominantly affect the nucleus, and to preferentially involve non-neuronal cells.

Relative lack of toxicity of transplatin compared with cisplatin in rodents.

The differential toxicity of the cis- and trans-isomers of diamminedichloroplatinum (II) (cisplatin and transplatin) was investigated in rats and guinea pigs. In both species, repeated daily administration of 1 to 2 mg per kg cisplatin produced severe histological and/or functional damage to renal and gastro-intestinal systems and resulted in death of the animals. Quantification of tissue platinum by atomic absorption spectroscopy demonstrated accumulation of large amounts of platinum in the kidney of the animals, with lesser amounts in the liver and gastro-intestinal tract. Transplatin, administered at total doses two- to four-fold that of cisplatin, was essentially non-toxic by histological and functional assessment. However, the amounts of tissue platinum measured in transplatin-treated animals were no smaller than those measured in cisplatin-treated animals; indeed, platinum concentrations in kidneys of transplatin-treated rats were more than 2.5 times those in cisplatin-treated rats. Thus tissue platinum content did not correlate with organ damage. These data suggest that mechanism(s) involving steric interactions of platinum species, perhaps with cellular macromolecules such as DNA or RNA, may be important in the differential toxicity of these two compounds.

Toxicity of cis-diamminedichloroplatinum (II) (cisplatin) in the frog, Rana pipiens.

The toxicity of the anti-cancer drug cis-diamminedichloroplatinum (II) (cisplatin), at 2 to 40 mg per kg, was studied in the frog, Rana pipiens. The LD50 for the drug was approximately 17 mg per kg. Major non-nervous system toxicity occurred in the kidney. Renal failure was manifested as anasarca and increasing blood urea nitrogen (BUN). Histopathological changes included acute tubular necrosis and tubular dilatation, which were more severe at higher doses. Interstitial fibrosis occurred after prolonged survival after a single dose. Ultrastructurally, there was increased electron-dense material in tubular cells, but no specific changes in mitochondria or nuclear structures were seen. Gastro-intestinal toxicity was less severe than in other species and was more prominent at higher doses. Pathological changes consisted of epithelial nuclear atypia and apoptosis. By electron microscopy, there was increased separation of cell borders, depletion of chylomicrons and mucin granules and increases in electron-dense material. Again no specific mitochondrial or nuclear changes were seen. Relatively slight changes were seen in the liver, consisting of altered distribution of rough endoplasmic reticulum and dispersion of nuclear chromatin. Minimal pathology was demonstrated in the haematopoietic system or in the gonads. Thus toxicity of cisplatin in the frog is similar to that seen in mammals, including rodents and man.

Cisplatin-induced neurotoxicity with seizures in frogs.

Cis-diamminedichloroplatinum II (cisplatin) given by injection to adult frogs (Rana pipiens) resulted in tonic-clonic seizures 3 to 5 weeks later. The seizures could be induced multiple times; the animals appeared entirely normal between seizures. In the spinal cord there was vacuolation in the anterior grey horns. Ultrastructurally, the vacuoles consisted of swollen astrocytic processes in the neuropil and around neurons. Generalized edema with swelling of perivascular astrocyte foot processes was not seen. Systemic administration of cisplatin to frogs results in neurotoxicity with seizures and astrocytic swelling in the spinal cord.