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PubMed1995

Neurologic evaluation of the rat during sciatic nerve block with lidocaine.

BACKGROUND: Quantitative behavioral testing is necessary to establish a reproducible measure of differential functional blockade during regional anesthesia. Methods for assessment of the neurologic status (mental status, posture, gait, proprioception, motor function, autonomic function, and nociception) in veterinary neurology were adapted for the rat and used to monitor functional changes separately during a sciatic nerve block. METHODS: Sprague-Dawley rats were acclimated to laboratory routine before the study so that lidocaine (0.1 ml, 1%) could be injected near the sciatic notch without any chemical restraint. The onset, duration, and magnitude of functional losses were monitored. Proprioceptive integrity was evaluated by assessing the response to tactile placing and the hopping response. Extensor postural thrust, a test for postural reactions in small animals, was assessed on a digital balance and found adequate for quantifying motor function. Analgesia was assessed by measuring withdrawal response latencies to noxious thermal stimulation (51 degrees C) and to superficial and deep noxious pinches. Autonomic function was monitored by measuring skin temperature. Contralateral limb function was used as an internal control, and injection of saline was used as an external control in separate, control animals. RESULTS: Onset of postural and gait abnormalities were observed as early as 40 s after injection. On each occasion proprioceptive impairment was detected first, followed by impairment of motor function and nociception. Complete absence of proprioception occurred from 10 to 30 min (n = 9) and of motor function at 30 min after injection (n = 10); both functions were fully recovered by 120 min. A unilateral increase in skin temperature on the foot was detected by 1 min; had reached its maximum change, 5.3 +/- 0.7 degrees C, at 10 min; and had returned to control levels at 60 min after injection (n = 12). Withdrawal response to cutaneous or superficial pain was absent in all ten animals from 5 to 30 min whereas the response to deep pain was absent in all ten animals at 20 min only. The response to noxious stimulation recovered at 90 min. Attention was paid to the temporal relation of the impairment of various functions. CONCLUSIONS: Quantitative observations of the onset, offset, and intensity of differential functional impairment or block over time will make it possible to establish the doses and conditions for local anesthetics that result in differential nerve block and will permit comparison of these changes among different drugs and "clinical" protocols.

Animals↗
PubMed1990

Modulation of constitutive cytochrome P-450 expression in vivo and in vitro in murine keratinocytes as a function of differentiation and extracellular Ca2+ concentration.

A procedure was developed for the per cell estimation of cytochrome P-450-dependent monooxygenase activities in cultures and whole cell suspensions of murine epidermal keratinocytes (MEKs). Murine keratinocytes cultured in medium containing less than or equal to 0.04 mM Ca2+ can be induced to differentiate by raising medium Ca2+ concentrations to 1.2 mM. The per cell activities of the monooxygenases 7-ethoxyresorufin O-deethylase (7-ER) and 7-ethoxycoumarin O-deethylase (7-EC) were elevated greater than or equal to 2090% and approximately 460%, respectively, within 13-24 hr of Ca2+ shift. These increases could be completely suppressed by supplementation of culture medium with actinomycin D or cycloheximide immediately prior to Ca2+ shift. After prolonged culture in low Ca2+ medium, some MEKs detached from the monolayer. These detached cells had the characteristics of differentiating MEKs but did not have elevated 7-EC or 7-ER activities. Percoll gradient centrifugation of freshly isolated dorsal skin MEKs was used to prepare four subpopulations that differed in their stages of terminal differentiation. 7-EC and 7-ER activities varied among these subpopulations and correlated with the degree of MEK differentiation. Specifically, the lowest and highest per cell activities (greater than 7-fold difference) were in the basal and most differentiated spinous cell populations, respectively. Collectively, the current studies demonstrate that in vivo P-450 activities are markedly different in proliferating and differentiating MEKs and suggest that constitutive P-450 expression may be modulated as a function of changes in Ca2+ concentration that occur during keratinocyte terminal differentiation.

Animals↗
PubMed2003

Rapid formation of hepatic organoid in collagen sponge by rat small hepatocytes and hepatic nonparenchymal cells.

BACKGROUND/AIMS: Hybrid bioartificial liver devices supporting a large mass of metabolically active hepatocytes are thought to be necessary for the successful treatment of patients with severe acute liver failure. However, it is very difficult to obtain cells with both growth activity and differentiated functions. Rat small hepatocytes (SHs), which are hepatic progenitor cells, can differentiate into mature hepatocytes and reconstruct a hepatic organoid by interacting with hepatic nonparenchymal cells (NPCs). METHODS: Colonies of SHs were collected and replated on a collagen sponge. Hepatic functions were examined by ELISA, immunoblotting, and Northern blotting. Cells in the sponge were characterized by immunocytochemistry and transmission electron microscopy. Urea synthesis was measured and metabolization of fluorescein diacetate was examined. RESULTS: SHs could proliferate and expand to form a hepatic organoid in the sponge. Albumin secretion and other hepatic protein production of the cells in the sponge increased with time in culture and the amounts were much larger than for those obtained from cells grown on dishes. Morphologically and functionally differentiated hepatocytes were observed and some CK19-positive cells formed duct-like structures within the sponge. Excretion of fluorescein was observed in bile canaliculi. CONCLUSIONS: Hepatic organoids can be rapidly reconstructed in a collagen sponge by rat SHs and NPCs.

Animals↗
PubMed1989

The onset and progression of osteoblast differentiation is functionally related to cellular proliferation.

The relationship of proliferation to the developmental sequence associated with bone cell differentiation was examined in primary osteoblast cultures derived from fetal rat and embryonic chick calvaria. A reciprocal and functional relationship exists between the decline in proliferative activity which occurs during the initial stages of the developmental sequence and the induction of genes encoding osteoblast phenotype proteins associated with matrix maturation and mineralization. This relationship is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase (AP) and osteopontin (OP) genes immediately following the proliferative period and expression of osteocalcin with the onset of mineralization, and 2) increases in AP and OP when DNA synthesis is inhibited. By determining cellular mRNA levels and rates of mRNA synthesis in isolated nuclei, we found that the down-regulation of cell growth-related genes is modified at both the levels of transcription and mRNA stability. For a histone gene where down-regulation is transcriptionally mediated, we have observed that the shutdown of osteoblast proliferation is associated with the selective loss of the interaction of a promoter binding factor (HiNF-D) with a proximal regulatory element (Site II). A relationship between Site II occupancy by HiNF-D and the onset of osteoblast differentiation is supported by the persistence of Site II-HiNF-D interactions when proliferating rat osteoblasts are growth arrested under conditions that do not induce differentiation; and additionally, by the loss of Site II-HiNF-D interactions during the shut-down of proliferation when HL60 promyelocytic leukemia cells are induced to differentiate into monocytes. Our results are consistent with a requirement of proliferation for expression of genes involved with production, deposition and possibly organization of the osteoblast extracellular matrix. It is also reasonable to postulate that properties of the mineralizing matrix are related to the shut-down of proliferation.

Animals↗
PubMed1995

Function and differential regulation of the alpha 6 integrin isoforms during parietal endoderm differentiation.

F9 embryonal carcinoma cells treated with retinoic acid differentiate in monolayer into parietal endoderm (PE) or in suspension into embryoid bodies with an outer layer of visceral endoderm (VE) surrounding a core of largely undifferentiated cells. Previous reports have shown that cell-extracellular matrix interactions mediated by the beta 1 integrins play a critical role in the differentiation and migration of PE. In the present study we investigated the pattern of expression and function of the integrin alpha 6 beta 1 during the differentiation of F9 cells into VE and PE. F9 cells express integrin subunits alpha 3, alpha 5, alpha 6, and beta 1. Cell adhesion and migration assays demonstrate that alpha 6 beta 1 is the major laminin receptor in undifferentiated F9 cells as well as F9-derived PE cells. However, the amount of alpha 6 protein decreases significantly upon F9 cell differentiation into either VE or PE, as revealed by immunofluorescent staining and immunoprecipitation analysis. In contrast, the amount of steady-state alpha 6 message stays constant before and after F9 cell differentiation, suggesting that the down-regulation of alpha 6 beta 1 occurs post-transcriptionally. In view of previous reports of two alpha 6 isoforms generated by alternative RNA processing, we carried out reverse transcription-PCR analysis and show that, while alpha 6B is the major mRNA isoform before and after F9 cell differentiation, alpha 6A mRNA is weakly expressed in undifferentiated F9 cells and is substantially increased following F9 differentiation into PE. Immunoprecipitations using the isoform-specific antibodies show an increase in alpha 6A and a dramatic decrease in alpha 6B protein following PE differentiation. Pulse-chase experiments indicate that, whereas the stability of alpha 6B protein is unaltered, synthesis of alpha 6B protein is decreased at least threefold following PE differentiation. Further experiments demonstrate that alpha 6A localizes to focal contacts in PE cells. The switch from alpha 6B to alpha 6A and the localization of alpha 6A at focal contacts correlate with the acquisition of PE cell motility, which suggests distinct functions for the two alpha 6 isoforms.

Animals↗
PubMed2003

Inducibility of microsomal liver function may differentiate cirrhotic patients with maintained compared with severely compromised liver reserve.

BACKGROUND AND AIM: Quantitative tests of liver function (QTLF) can be modulated by enzyme-inducing agents. The objective of the study was to examine changes in QTLF after treatment with Phenobarbital, a potent cytochrome P450-inducing agent. METHODS: Forty-six consecutive patients with liver cirrhosis Child-Pugh score B and C (34 alcoholic, 12 hepatitis C) and a compromised aminopyrine breath test (ABT) were included. Thirty-six patients (group I) received phenobarbitone (150 mg) for 7 days. Ten patients (group II) received a placebo. The QTLF, which included ABT (microsomal liver function), galactose elimination capacity (GEC, cytosolic liver function), sorbitol clearance (SCl, liver plasma flow) and indocyanine green clearance (ICG, liver perfusion) was carried out before and after pharmacological induction. RESULTS: Group I showed a basal ABT of 0.18 +/- 0.11% dose.kg/mmol CO2 (normal >0.6%), which increased significantly after induction (172%, P < 0.05), whereas in group II the ABT values did not change. In group IB, a subgroup of group I which exceeded the basal threshold value of 0.30% after stimulation (n = 22), the ABT values increased significantly to 0.44 +/- 0.17% (244%, P < 0.01). The GEC, SCl and ICG remained unchanged before and after induction. CONCLUSIONS: After pharmacological induction, microsomal liver function increased significantly in a subgroup of patients with liver cirrhosis, whereas the GEC, SCl and ICG remained unchanged. Inducibility of the microsomal liver function could be used as a novel parameter of functional hepatic reserve and prognosis in cirrhosis.

Adult↗
PubMed1985

1,25-Dihydroxyvitamin D3 and the regulation of the differentiation and function of macrophages and granulocytes.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) exerts a differential inhibitory effect on the formation of granulocyte, granulocyte/macrophage, and macrophage colonies grown from mouse bone marrow precursor cells; 50% inhibition was attained at 1.1, 2.3, and 23 nM 1,25(OH)2D3, respectively. The inhibition of colony formation, as well as phagocyte proliferation in liquid cultures, requires the presence of 1,25(OH)2D3 in the early stages of culture (up to 72 h after culture initiation). 1,25(OH)2D3 induces a dose- and time-dependent augmentation of the phagocytic capability of mononuclear phagocytes (up to 100%) towards both heat-killed yeast cells and IgG-coated sheep red blood cells. The augmentation of the phagocytic capability of the mononuclear phagocytes depends critically on when 1,25(OH)2D3 is added. It is effective when added up to 72 h after culture initiation, while at later stages (greater than or equal to 96 h) the cells are no longer induced to express enhanced phagocytic capability. We suggest that these phenomena may be relevant to hemopoietic processes.

Animals↗
PubMed1998

Expression and effect of insulin-like growth factor I on rat fetal Leydig cell function and differentiation.

Insulin like growth factor I (IGF-I) is believed to be a potent para/autocrine stimulator of Leydig cell function in adult testis. We investigated whether IGF-I is also an intratesticular regulator of fetal Leydig cell function by measuring its production in the fetal testis and its ability to affect testicular steroidogenesis during fetal development. Northern blot analysis revealed one major IGF-I transcript of 7-7.5 kb and two minor transcripts of 3.8 and 1.8 kb in 20.5 day fetal testis. IGF-I was detected by RIA in 16.5 fetal day testes, and the amounts of IGF-I secreted by 16.5 and 20.5 fetal day testes in vitro were much greater than the amounts contained in the testes, indicating active synthesis in culture. The secretion of IGF-I by the fetal testis in vitro was increased with testicular age and time in culture. It was not modified by gonadotropins or (Bu)2cAMP. Testosterone secretion by fetal testes explanted 13.5, 16.5, 18.5, and 20.5 days after conception and cultured in the presence or absence of 100 ng/ml LH for 3 days was not affected by the addition of 50 ng/ml IGF-I to the medium. In contrast, the addition of IGF-I to dispersed fetal testicular cells cultured for 3 days in the presence or absence of LH increased the number of Leydig cells identified by a positive cytochemical reaction for 3beta-hydroxysteroid dehydrogenase (3betaHSD). This was more pronounced with cells from 16.5- day-old fetuses (stage when the fetal Leydig cells are differentiating in vivo) than with 20.5-day-old fetuses cells (stage when the number and the function of fetal Leydig cells are stable or decreasing). It results from both an increased differentiation of mesenchymal cells in fetal Leydig cells and an increase in the mitotic index of the fetal Leydig cells, as inferred from the small increase in the percentage of bromodeoxyuridine/3betaHSD-positive cells. Both LH and IGF-I increased significantly testosterone production by day 16.5 cells. In the presence of LH, a high amount of testosterone was produced per 3betaHSD-positive cell; IGF-I further increased this production. This effect was not observed with day 20.5 cells. The amounts of testosterone produced per 3betaHSD-positive cell cultured in the presence of both LH and IGF-I were more than additive. Like IGF-I, insulin (50 ng/ml) increased testosterone secretion per 3betaHSD-positive cells in cultures of day 16.5 cells, but not in those of day 20.5, cells. Lastly, IGF-I also increased the steroidogenic activity of each Leydig cell in cultures containing (Bu)2cAMP, but its effects were weaker than those observed in the presence of LH. This suggests that IGF-I has sites of action both upstream and downstream cAMP generation. These results suggest that IGF-I acts as paracrine/autocrine factor in the differentiation and activity of fetal Leydig cells.

Animals↗
PubMed2005

Growth restriction and exendin 4 promote endocrine expression in cultured islet cells derived from patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI).

Islets derived from patients with persistent hyperinsulinemic hypoglycemia of infancy, PHHI, show a significant capacity to proliferate in vitro without the addition of growth factors. However, as with other differentiated cells, PHHI-derived islet cells show a loss of differentiated function with repeated subculture. Here, we have investigated methods of extending the differentiated function of PHHI-derived endocrine cells following in vitro expansion. The experiments were carried out on 13 primary pancreatic cell cultures from patients with PHHI, the majority of which (n = 11) were glucose unresponsive--a distinctive feature of PHHI disease. After a 20-day period of cell expansion in 10% FCS, cells were switched to media containing varying concentrations of FCS with or without exendin 4 and endocrine function was measured using ELISA and RT-PCR for insulin and PDX-1. Switching the expanded cultures to low serum was shown to slow cell division while maintaining the residual differentiated endocrine characteristics of all the cultures tested. Exendin 4 was shown to further enhance the improved insulin secretion shown by low serum cultures, although in glucose nonresponsive cells, this was at the expense of insulin stores. However, we did observe that exendin 4 could upregulate insulin secretion, insulin storage, and PDX-1 expression in glucose responsive PHHI cultures.

Cell Proliferation↗
PubMed1990

Natural history of pelviureteric obstruction detected by prenatal sonography.

Fifty-six infants with prenatally diagnosed hydronephrosis are reported. In all instances the diagnosis was confirmed postnatally and at renography 45 were obstructed; 38 obstructed kidneys (84%) and all of the non-obstructed kidneys had a differential function exceeding 40% of total function. Latterly we have come to recommend early pyeloplasty only if differential function of a renographically obstructed kidney is less than 40%; 6 early pyeloplasties were performed for this reason; 28 infants (30 renal units) were managed non-operatively and 18 of these (19 renal units) were reassessed renographically. In 11 the obstruction persists. Sonography demonstrated improving hydronephrosis in 8 kidneys with resolution in 5 and no change in 6. Of the other 10 infants (11 renal units), the hydronephrosis has improved in 4, resolved in 1 and remains unchanged in 6. Our experience suggests that neonatal and early pyeloplasty can be restricted to a modest number of infants in whom there is impaired renal function. In cases with normal function the natural history appears essentially benign and does not justify routine pyeloplasty.

Female↗
PubMed1988

In vitro toxicology: a commercial proposition?

1. The commercial value of any test system must be equated with its scientific value. In vitro toxicity tests only have scientific value when they are accurate models of toxicity in vivo. 2. Toxicity is a complex event so that the simple tests of cell viability are unlikely to be useful models as these tests often use cell lines which are functionally remote from cells in tissues of the whole animal. 3. Primary cultures retain their differentiated function for hours (hepatocytes) or days (nerve cells) and may acquire differentiated functions in vitro (embryo cells) and are the models of choice for predicting toxic hazard. 4. When the in vitro test is a satisfactory predictive model of toxicity there are sound commercial reasons for its use. It saves time and effort and can be used at an early stage in compound development to sort out the toxic from the non-toxic candidate compounds. Large numbers of structurally similar compounds can be tested (QSAR studies) and a greater understanding of the mechanism of toxic action can often be achieved than in vivo because of the numbers of animals required and the interplay of different organ systems in the toxic events that make interpretation difficult. Thus the in vitro test can not only save animals but can also save compounds.

Animals↗
PubMed2003

Pituitary hormones inhibit the function and differentiation of fetal Sertoli cells.

Although the role of pituitary hormones in fetal Sertoli cell proliferation is well understood, their involvement in fetal Sertoli cell differentiation is poorly documented. In this study, we evaluated rat fetal Sertoli cell function by measuring basal transferrin secretion ex vivo and transferrin and anti-Müllerian hormone (AMH) mRNA levels in vivo. The differentiation state of the Sertoli cells was estimated from the amount of transferrin secreted ex vivo after acute stimulation with FSH. Surprisingly, we found that the amount of transferrin secreted by each Sertoli cell in basal condition and after acute FSH stimulation decreased between 18.5 and 21.5 day post coitum (dpc), which corresponds to the onset of pituitary hormone secretion. All of the Sertoli cell parameters measured (basal and FSH-stimulated transferrin secretion ex vivo, transferrin and AMH mRNA levels in vivo) were higher in 21.5-dpc fetuses that had been decapitated on 16.5 dpc than in control littermates. Furthermore, immunostaining for AMH was strongly increased after decapitation. Taken together, these results suggest that pituitary hormones in the fetus and in the immature or adult rat differently regulate Sertoli cells, which suggests that fetal Sertoli cells have their own particular physiology.

Animals↗