Regulation of the perilymphatic-endolymphatic water shunt in the cochlea by membrane translocation of aquaporin-5.

Volume homeostasis of the cochlear endolymph depends on radial and longitudinal endolymph movements (LEMs). LEMs measured in vivo have been exclusively recognized under physiologically challenging conditions, such as experimentally induced alterations of perilymph osmolarity or endolymph volume. The regulatory mechanisms that adjust LEMs to the physiological requirements of endolymph volume homeostasis remain unknown. Here, we describe the formation of an aquaporin (AQP)-based "water shunt" during the postnatal development of the mouse cochlea and its regulation by different triggers. The final complementary expression pattern of AQP5 (apical membrane) and AQP4 (basolateral membrane) in outer sulcus cells (OSCs) of the cochlear apex is acquired at the onset of hearing function (postnatal day (p)8-p12). In vitro, hyperosmolar perfusion of the perilymphatic fluid spaces or the administration of the muscarinic agonist pilocarpine in cochlear explants (p14) induced the translocation of AQP5 channel proteins into the apical membranes of OSCs. AQP5 membrane translocation was blocked by the muscarinic antagonist atropine. The muscarinic M3 acetylcholine (ACh) receptor (M3R) was identified in murine OSCs via mRNA expression, immunolabeling, and in vitro binding studies using an M3R-specific fluorescent ligand. Finally, the water shunt elements AQP4, AQP5, and M3R were also demonstrated in OSCs of the human cochlea. The regulation of the AQP4/AQP5 water shunt in OSCs of the cochlear apex provides a molecular basis for regulated endolymphatic volume homeostasis. Moreover, its dysregulation or disruption may have pathophysiologic implications for clinical conditions related to endolymphatic hydrops, such as Ménière's disease.

All functional aquaporin-4 isoforms are expressed in the rat cochlea and contribute to the formation of orthogonal arrays of particles.

The water channel aquaporin-4 (AQP4) is expressed in the cochlea and is essential for normal hearing. Unlike other AQPs, multiple isoforms of AQP4 have been reported in diverse tissues, three of which, M1, M23, and Mz, function as water channels. In addition, these protein isoforms are found in higher order complexes. Morphologically these higher order complexes correspond to orthogonal arrays of particles (OAPs) that are found in cell membranes by freeze fracture analysis. Using RT-PCR, quantitative PCR and blue-native PAGE immunoblots we identified all functional AQP4 isoforms -M1, M23, and Mz- and the formation of higher-order complexes in the organ of Corti of the rat. Complementary freeze-fracture studies revealed OAPs distributed in the lateral and basal membrane domains of the cochlear duct supporting cells, specifically Hensen's cells and outer sulcus cells. The unique inter- and intracellular heterogeneity in size, density and shape of OAPs suggests exceptional physiological requirements for the maintenance of water homeostasis during auditory sensory transduction in the cochlea.

Spontaneous pathology of the gray short-tailed opossum (Monodelphis domestica).

The gray short-tailed opossum, Monodelphis domestica, is a newly established laboratory animal that is becoming increasingly important to biomedical research. Because little disease information is available for this species, we reviewed records for spontaneous gross and histologic lesions and microbiologic results in 150 M. domestica necropsies during an 11-year period. We identified 150 (91 female, 59 male) animals from 441 necropsy cases which were controls in experimental protocols or were members of the breeding colony. Initial statistical examinations indicated that the sample was representative of the living members of the breeding colony with respect to age, sex, and range of inbreeding. Causes of death and types of tumors were specifically evaluated. Females died earlier than males (22.6 +/- 13.0 months versus 30.9 +/- 11.9 months), but this difference was not associated readily with diet or inbreeding levels. The organ systems with the greatest lesion prevalences, in decreasing order, were the digestive, urogenital, cardiovascular, and respiratory systems. The most probable causes of all deaths were associated with the digestive system, followed by the cardiovascular and integumentary systems. The principal disease problems were rectal prolapse, congestive heart failure, and dermatitis. Neoplasia was found in 39 of the animals. The prevalence of neoplasia was greatest in the digestive system, followed by the endocrine, urogenital, integumentary, and hematopoietic systems. Pituitary adenoma was the most common neoplasm, followed by uterine leiomyoma and cutaneous lipoma. Specific microbially-induced diseases were not recognized, and endo- and ectoparasites were not found in colony-born M. domestica.

Dietary modulation of the progression of nephropathy in aging rats: an evaluation of the importance of protein.

Chronic nephropathy involving glomerular sclerosis markedly progresses in severity with age in male Fischer 344 rats fed ad libitum. Restricting food intake by 40% almost totally prevents progression of these lesions. Restricting food intake by 40% without restricting protein intake is also highly effective although somewhat less so than food restriction that includes protein restriction. These findings indicate that reducing the intake of protein is not the major reason for the retardation by food restriction of the age-associated progression of nephropathy in rats.

Influence of the restriction of individual dietary components on longevity and age-related disease of Fischer rats: the fat component and the mineral component.

The influence of restricting either the fat or the mineral component of the diet to the same extent as they are restricted in the life-prolonging, food-restriction paradigm but without restricting calories was studied in regard to longevity and age-related pathologic lesions of barrier-maintained male Fischer 344 rats. Neither the restriction of fat nor the restriction of mineral influenced the median length of life or maximum life span as indicated by the age of the 10th percentile survivors. Restricting the dietary fat did retard the development of chronic nephropathy and associated lesions, but it also increased the prevalence of lymphoma and leukemia. The development of chronic nephropathy was not significantly affected by restricting the mineral component of the diet.

The influence of dietary protein source on longevity and age-related disease processes of Fischer rats.

The influence of replacing dietary casein with soy protein on longevity and age-related pathologic lesions of male Fischer 344 rats was investigated. Caloric intake and body weights were similar for rats on the two diets. Rats on the soy protein-containing diet had a median length of life of 844 days compared to 730 days for those on the casein-containing diet (p less than .002), and the ages of the 10th percentile survivors were 937 and 857 days, respectively (p less than .02). The progression of chronic nephropathy was markedly retarded by replacing casein with soy protein. Only 7% of the rats dying spontaneously on the soy protein-containing diet exhibited end-stage chronic nephropathy compared to 41% of the rats on the casein-containing diet. Clearly, the soy protein-containing diet enables ad libitum fed male Fischer 344 rats to be used as a model for aging research without the occurrence of renal failure as a major confounding problem.

Case of disseminated African histoplasmosis in a baboon.

An adult red baboon (Papio cynocephalus papio) had a severe disseminated case of African histoplasmosis (Histoplasma capsulatum var. duboisii) with skin lesions from small pustules to ulcerative granulomas. Osteolytic lesions were seen radiographically. Treatment with ketoconazole for 6 months was unsuccessful.

Pathophysiology of soman intoxication in primates.

Adult baboons were monitored during intravenous infusion of Soman (1,2,2-trimethylpropyl ester, phosphonofluoridate). Three groups of animals were studied. Two groups were anesthetized with sodium pentobarbital (initial dose, 20 mg/kg), instrumented for measurement of systemic blood pressure (BP), pulmonary artery pressure, cardiac output (CO), ECG, ventilatory flow, translaryngeal pressure (PTL), transdiaphragmatic pressure (Pdi), transpulmonary pressure (PTP), diaphragm EMG, and efferent phrenic nerve traffic (Eph). One group received no Soman and served as controls. In the other group, Soman was infused over 10 min at doses of 13.1, 8.21, 4.92, or 3.3 micrograms/kg. The onset of intoxication occurred within 7-8 min (before the end of the 10-min infusion), manifested by muscular fasciculations, stridorous breathing, copious secretions, and atrioventricular arrhythmias. Mean BP decreased to 30 mm Hg by the combination of decreased CO and decreased vascular resistance. There was a dose-related response in the onset and duration of these effects. Apnea occurred in most animals and coincided with cessation of the Eph signal. Stimulation of the diaphragm via the phrenic nerve following apnea yielded Pdi values unchanged from baseline, indicating an intact neuromuscular apparatus. All animals required ventilatory support. Some surviving animals exhibited severe behavior changes. The third group of animals was studied without anesthesia. Instrumentation was performed 3 days before using a tether system for the measurement of BP, CO, and ECG, and an arterial line for blood withdrawal. Soman was infused over 10 min at a dose of 13.1 micrograms/kg. The onset of intoxication occurred within 2-3 min, manifested by hyperactivity, severe muscle fasciculations which simulated grand-mal convulsions, stridorous respiratory sounds, copious secretions, and cardiac arrhythmias. Apnea and severe lactic acid acidosis developed in all animals and all required ventilatory assistance. None recovered spontaneous ventilation at the end of 4 hr.

The aging AXC/SSh rat: assessment of longevity and prevalence of neoplastic and nonneoplastic diseases in necropsied rats.

Median life span, 906 days, was identical for conventionally reared, ad libitum fed AXC/SSh male and female rats and exceeded that of most other conventionally or barrier maintained rats. AXC/SSh rat longevity partially reflects the 25% incidence of moderate to severe nephropathy occurring at 30 to 41 months. Because male survival was related to sibship, p less than .02, and female survival was not, p greater than .05, a genetic component may affect male longevity. Maximum body weight was achieved during the initial 20% of life span and maintained through the final 10% when mean loss was 14 and 15%, respectively, for males and females. Pituitary adenomas occurred in 65% of females and 10% of males. Mammary adenocarcinoma occurred in 26% of females. Interstitial cell tumors were not detected in males younger than 24 months. Prostate adenocarcinomas occurred in 23% of males older than 29 months. The longevity of AXC/SSh rats and their low incidence of nephropathy suggests they are an attractive model for studies in aging.

Selected coagulation reference values for adult and juvenile baboons.

Sodium citrated blood samples were collected from clinically normal baboons of various ages. Prothrombin times, activated partial thromboplastin times and quantitative fibrinogen levels were determined using current, widely available techniques. Arithmetic mean values and observed ranges were determined for 61 animals.

Nutritional influences on aging of Fischer 344 rats: II. Pathology.

The aim of this study was to explore the effects of nutritional manipulations on the occurrence and progression of age-related pathologic lesions in male Fischer 344 rats. The following nutritional regimens were studied: (a) ad libitum feeding, (b) food restriction initiated at 6 weeks of age, (c) food restriction initiated at 6 months of age, (d) food restriction limited to a period of early life (6 weeks to 6 months of age), (e) protein restriction without caloric restriction. The major age-related lesions observed were chronic nephropathy, cardiomyopathy, and neoplasia. Food restriction initiated at 6 months of age was as effective as food restriction initiated at 6 weeks of age in slowing the progression of chronic nephropathy and cardiomyopathy and in delaying the occurrence of neoplasia. Food restriction limited to early life was much less effective. Protein restriction in the absence of caloric restriction did not delay the occurrence of neoplasia, but it did retard chronic nephropathy and cardiomyopathy, although much less effectively than caloric restriction involving a similar level of protein restriction.

Gastric stapling for morbid obesity: gastrointestinal response in a rat model.

The adaptive response of the gastrointestinal tract to gastric stapling was studied in a Zucker (fafa) genetically obese rat model. The effects of gastric stapling in rats with a Roux-en-Y gastrojejunostomy were compared to Roux-en-Y and intact controls. Rats (225 to 275 g) were divided into three groups: group I (GI), Roux-en-Y, stapled, ad libitum fed; group II, Roux-en-Y, unstapled; and group III, intact (laparotomy only). Groups II and III were further subdivided into group IIA (GIIA) and group IIIA (GIIIA), pair-fed to GI; and group IIB (GIIB) and Group IIIB (GIIIB), fed ad libitum. All rats were fed a diet of liquid rat formula and rat food (50:50 cal ratio). After 2 wk rats were killed and the liver, pancreas, and stomach removed, weighed, and tissue taken for histology. The intestine was divided into three segments corresponding to the excluded segment (1, duodenum), transposed segment (2, jejunum), and ileal segment (3), with tissue taken for histology. Liver lipid, pancreatic amylase, serum amylase and gastrin, and intestinal mucosal protein and DNA were measured. Weight gain, g/14 days, was: GI, 24.1 +/- 7.8; GIIA, 17.0 +/- 2.0; GIIB, 42.6 +/- 4.8; GIIIA, 17.6 +/- 4.7; GIIIB, 54.6 +/- 8.9. All rats were in positive N balance. Liver weight and lipid were similar in all groups. Pancreatic mass was significantly increased in all Roux-en-Y animals, while amylase activity per g tissue was significantly less than in intact animals. Stapled rats had atrophy of the glandular portion of the stomach, decreased stomach mucosal weight, and lower serum gastrin concentration compared with all other rats (p less than 0.01). In stapled rats, segment weight and mucosal weight were lower in the excluded segment, and higher in the transposed segment, compared to all other rats. Tissue pathology was found only in stapled rats: gastric atrophy, focal fibrosis of gastric wall, peritonitis, minimal to mild multifocal hepatitis, and mild periductal fibrosis of the pancreas. There are significant early adaptive and pathological changes after gastric stapling in the rat.

Nasal polyposis in a chimpanzee.

A 15-year-old female chimpanzee with nasal polyposis sustained respiratory compromise when she was sedated and expired despite resuscitative efforts. Postmortem examination revealed very large fibromyxomatous nasal polyps completely obstructing the upper airway. Gross examination and histopathologic findings were indistinguishable from those of human polyps. The chimpanzee is a potential animal model for nasal polyposis that could provide basic information concerning the relationship of polyps to type I hypersensitivity and to three severe respiratory tract disorders in humans: cystic fibrosis, bronchial asthma, and the immotile cilia syndrome.

Malignant lymphoma and Hodgkin's disease in baboons (Papio sp.).

Four cases of spontaneous malignant lymphoma and one of Hodgkin's lymphoma in baboons at the Southwest Foundation for Biomedical Research were studied and described. These cases were in animals of both sexes that varied in age from 6 to 25 years, and were in residence at the Foundation from 2 to 24 years, during which time there was no known exposure to carcinogenetic agents. Attempts to isolate an etiological viral agent or demonstrate viral particles in lymphoid tissue were unsuccessful.

Histologic classification as an indication of therapeutic response in malignant lymphoma of dogs.

In retrospective evaluation of treatment of canine malignant lymphoma, 12 of 13 dogs that had received doxorubicin alone or in combination with dacarbazine attained complete remission. Doxorubicin had been given alone, with combination chemotherapy being used only when complete remission could not be achieved and maintained with doxorubicin. The response to single or combined chemotherapy was correlated with histologic cell type of the malignant lymphoma. Histiocytic cell types did not respond to doxorubicin alone, but lymphoblastic types did respond. Combination chemotherapy was effective against histiocytic types. A mixed-cell type, which was initially responsive to doxorubicin alone, but not responsive after relapse, was observed to be histiocytic on rebiopsy.

Rescue by peripheral blood mononuclear cells in dogs from bone marrow failure after total-body irradiation.

In order to determine the minimum dose of buffy coat cells necessary to achieve hematopoietic rescue following supralethal irradiation, mongrel dogs under general anesthesia were subjected to leukacytapheresis using three different techniques of cell separation. The buffy coats were frozen with dimethylsulfoxide and stored at -196 degrees C until transfused. Sixteen dogs were irradiated with 800 rads and were supported with antibiotics and transfusions of irradiated homologous blood. They were transfused with the frozen and thawed buffy coat cells, and, if they survived, they were followed for 100 days, sacrificed, and their tissues studied. The mean yield of mononuclear cells during leukocytapheresis ranged from 4.1 +/- 2.0 X 10(9) (mean +/- SD) to 6.0 +/- 4.0 X 10(9) for the three leukacytapheresis methods; one technique was not as satisfactory as the other two. Six of the 16 dogs fully recovered with evidence of marrow rescue; however, only one had a dose of mononuclear cells less than 11.1 X 10(9). These data indicate that seven to 17 leukacytapheresis procedures would be required to reconstitute a 70 kilogram patient. These preliminary findings suggest that, because the yields of transplantable cells with current technology are not adequate, the transplantation potential of buffy coat cells exposed to mobilizing agents should be evaluated.