Ovarian effects of bovine follicular fluid treatment in sheep and cattle.
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Animals↗
Biomedical subjects
J K Critser
Publications and source records attributed to J K Critser.
Alteration in platelet count during early pregnancy in the mouse.
Published reports of pregnancy associated thrombocytopenia in mice have utilized the Quackenbush strain. The inability of some laboratories to verify this observation in other mouse strains prompted us to report our findings by using Swiss Albino ICR mice. In Exp. 1, pregnant and pseudopregnant mice were bled prior to mating (time 0) and daily on day 1 (vaginal plug) through day 7. In Exp. 2, media from 24 hr cultures of 2-cell mouse embryos or media from unfertilized oocytes were injected into splenectomized mice. Animals were bled at time 0 (before injection) and at 30, 60, and 120 min after injection. In Exp. 3, splenectomized mice were treated with either media from 2-cell stage embryos or with media supplemented with synthetic platelet-activating factor (PAF: 0.05, 0.1 or 0.2 micrograms). Animals were bled as in Exp. 2. Platelet numbers were determined in duplicate from each blood sample by using a hemacytometer. In Exp. 4, antagonist (SRI 63-441) or vehicle was administered to mated mice on days 1 through 4 of pregnancy. Animals were examined on day 8 to determine number of developing conceptuses. In Exp. 1-3, data were analyzed by using ANOVA for repeated measures, and in Exp. 4 data were analyzed by chi-square analysis. In Exp. 1, there was a treatment x time interaction (P less than .06) due to transient thrombocytopenia in pregnant but not pseudopregnant mice.(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of Variance↗
Evaluation of circulating anti-sperm antibodies in fertile and patient populations.
Several reports have demonstrated the presence of anti-sperm antibodies (ASA) in infertile populations; however there is a paucity of information regarding ASA in fertile populations. The purpose of this study was to establish objective criteria for the interpretation of the Immunobead Binding Test (IBT) based on values obtained from fertile individuals. Sera from 20 fertile couples (n = 40) were assayed by using a modification of the IBT previously described by Clarke et al. An initial lower limit of binding for positivity (lower limit) of 14% was used based upon the mean value for each isotype plus 2 standard deviations (SD) of 4 negative control sera assayed 4 to 7 times each. One-way ANOVA or chi-square analyses were used to analyze these data. There was no difference in percent immunobead binding between males and females in the fertile population (P greater than 0.1); therefore the data were pooled. Percent binding for fertile controls was: IgG, 21.7 + 31.9% (mean + SD); IgA, 19.5 + 25.8%; IgM, 16.9 + 14.9%. Initial analysis indicated no significant difference (P greater than 0.10) in percent binding between fertile and infertile individuals. The corresponding frequency of positive values (for at least one isotype) using a 14% lower limit was 23/40 (57.5%). This was not significantly different (P greater than 0.1) from the frequency observed in the patient population (140/242, 57.8%). New lower limits of positivity for each isotype were established based upon the mean plus 2 SD from the fertile control data: IgG, 85.4%; IgA, 71.1%; IgM, 46.7%.(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of Variance↗
Sperm antibodies after intraperitoneal insemination of sperm: a preliminary report.
To test the hypothesis that intraperitoneal insemination of sperm induces the expression of anti-sperm antibodies a prospective study was designed. Fifteen women undergoing intraperitoneal insemination (with or without oocyte transfer) were studied with 11 women having evaluation of anti-sperm antibodies. Sperm antibodies were detected by the immunobead test prior to intraperitoneal insemination and after each treatment cycle. Two criteria were used to assess positivity: the first based upon negative controls and the second based upon the evaluation of 20 fertile control couples. Using the first criteria, of 11 of the women undergoing IPI for the first time, 7 were initially negative and 4 were initially positive for at least 1 isotype. After treatment, 3 additional patients were positive (for a total of 7) and 4 patients remained negative. This alteration in sperm antibody frequency was not different (P = .4) as determined by the Fisher's Exact Test. Four of the 11 patients underwent a second cycle of IPI. All 4 patients were negative prior to the first treatment and 3 were negative prior to the second treatment. Subsequent to the second exposure, all 4 of these women were positive for at least 1 isotype. This shift in frequency distribution after 2 cycles was significant (P = .01). The frequency of antisperm antibodies for the same 11 patients was evaluated by using fertile control values as the basis of positivity. Two patients (18%) were positive for anti-sperm antibodies prior to intraperitoneal insemination. There was no change in the frequency of positivity after 1 cycle of IPI.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Determination of the osmotic characteristics of hamster pancreatic islets and isolated pancreatic islet cells.
The ability to store pancreatic islets using cryopreservation methodology would greatly assist the application of clinical islet transplantation to Type 1 (insulin-dependent) diabetics. It is our working thesis that the illumination of fundamental biophysical characteristics of these cells will lead to increased cryosurvival rates through theoretically predicted and experimental testing of optimal freezing protocol; as has been found for cells and tissues such as mammalian and Drosophila embryos. Pancreatic islets were isolated from Golden hamsters and their osmometric behavior, including inactive cell volume (Vb), was determined for either whole islets or isolated individual islet cells. When islets or islet cells were exposed to various concentrations of NaCl, they were found to exhibit a "classic" "Boyle-Van't Hoff" osmometric response. The Boyle-Van't Hoff representation of the volume curve (relative cell volume vs. 1/osmolality) yields a linear response with r values of .99 for each curve. Extrapolations to the normalized osmotically inactive volumes (Vb) were .43 and .22 for whole islets and individual islet cells, respectively. These data regarding the fundamental cryobiological characteristics of islets and islet cells should provide the foundation upon which to further the investigation of osmotic parameters of these cells and eventually lead to the determination of optimal freezing protocols.
Animals↗
Osmotic separation of pancreatic exocrine cells from crude islet cell preparations.
A novel approach is introduced here to selectively lyse exocrine cells in an islet preparation by hypo-osmotic treatment. Time to hypotonic cell lysis required for the islet cells was much longer than that for the exocrine cells, which permits a possibility of selectively killing the exocrine cells by hypotonic treatment. The first set of experiments was designed to select an appropriate osmolality for the hypotonic treatment. Kinetic changes in cell volume in response to extracellular anisosmolalities (30 to 90 mOsm/kg) were recorded using an electronic particle counter. The results indicated that, when exposed to a 30 mOsm/kg solution, islet cells swelled slowly to reach volumetric equilibrium in approximately 3 min. There was no significant hypotonic cell lysis observed even at the end of 4 min (n = 4). In contrast, pancreatic exocrine cells, when exposed to the same solution, expanded rapidly to the lytic volume and burst within 30 s. Significant exocrine cell lysis was invariably achieved within 30 s when cells were exposed to the osmolalities below 60 mOsm/kg. For osmolalities between 70 to 80 mOsm/kg, exocrine cell lysis was highly variable. When cells were exposed to 80 to 90 mOsm/kg, no significant cell lysis was observed. Thus, an osmolality of 50 mOsm/kg is recommended for hypotonic treatment, as it maximizes the lysis of exocrine cells without unnecessarily stressing (osmotically) the islet cells. The second set of experiments (time-course experiments, 20 to 120 s) was designed to determine the length of exposure time for which the exocrine cells were irreversibly damaged but the islet cells had only swollen to such a degree that cell function is restored upon returning to an isotonic condition. Viability of the hypotonic treated cells was evaluated at two different levels: membrane integrity, measured by combined fluorescent dye staining with propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), and mitochondrial function, measured by colorimetric MTT assay. The results showed that hypotonic treatment in a 50 mOsm/kg solution for 30 s resulted in over 85% loss of the membrane integrity for the exocrine cells. About 90% of these membrane lysed cells lost mitochondrial function (n = 3). By contrast, under the same treatment, less than 15% of the islet cells lost membrane integrity and mitochondrial function (n = 3). In conclusion, hypotonic treatment with a 50 mOsm/kg solution for 20 to 30 s at room temperature is sufficient to lyse the majority of the contaminating exocrine cells in an islet cell preparation, while maintaining function in the islet cells.
Animals↗
Hypoosmotic exposure of canine pancreatic digest as a means to purify islet tissue.
The development of more effective means to separate pancreatic islets from the unwanted exocrine tissue would greatly advance the field of clinical islet allotransplantation in the treatment of insulin-dependent diabetes mellitus. Recent experiments with hamster islets have demonstrated a selective destruction of dissociated single exocrine cells when exposed to hypotonic conditions. It was the aim of this study to extend these observations to the canine model with collagenase dissociated pancreatic tissue and to evaluate the treatment's effect on islet function. Pancreases from five mongrel dogs were digested using an automated protocol of intraductal delivery of collagenase, and gentle dissociation. Duplicate samples of pancreatic digest were removed for insulin and amylase determination prior to and immediately following exposure to 50 mOsm/kg salt solution for a period of 30, 60, or 300 s before returning the digest to isoosmotic conditions. The remaining digest was cultured for a period of 48 h at 37 degrees C before the tissue was recombined, washed, and a third sample removed for insulin and amylase. In vitro viability was then assessed using a static incubation assay with insulin content measured using a double-antibody radioimmunoassay, and amylase was determined using a colorimetric assay system. No difference in the insulin or amylase levels between the experimental groups was observed immediately following the hypotonic exposure; however, a significant decrease in the amylase content was observed following the 48-h culture period in digest that had been hypoosmotically exposed for 60 or 300 s compared with the pretreatment group (2.83 +/- 0.41 IU amylase/mg pancreas vs. 1.29 +/- 0.21 and 0.83 +/- 0.12, mean +/- SEM, p < 0.05). Insulin content was also significantly reduced in the 300-s exposure group compared with nontreated controls (3.2 +/- 0.6 mU insulin/mg pancreas vs. 2.0 +/- 0.2). The insulin/ amylase ratio (I/A), a measure of islet and exocrine content, was 1.1 +/- 0.13 following pancreas dissociation and 1.34 +/- 0.21 for control tissue cultured for 48 h. The I/A ratio increased following hypoosmotic exposure to 1.50 +/- 0.31 for tissue exposed for 30 s, 1.77 +/- 0.19 for 60-s exposure, and 2.54 +/- 0.13 for tissue exposed for 300 s (p < 0.05, vs. pretreatment group). In vitro insulin secretion was equivalent with the exception of the tissue exposed for 300 s, which had an increased basal level of insulin resulting in a significantly decreased stimulation index (3.8 +/- 0.5 vs. 8.1 +/- 1.2 for the purified islet control group, p < 0.05). These results suggest that a brief hypotonic exposure to pancreatic digest can alter the insulin/amylase ratio; however, there is a functional impairment on subsequent islet function after a period of in vitro tissue culture.
Animals↗
Osmotic tolerance limits of canine pancreatic islets.
Future improvements in the recovery and function of pancreatic islets following cryopreservation will require a more precise quantification of the stresses that occur at each stage of the cryopreservation protocol. Changes in solution osmolality during the addition and dilution of cryoprotectants and during freezing and thawing induce changes in islet volume that may exceed tolerable limits. The aim of this study was to determine the range of solution osmolalities that results in significant changes in islet function. Islets were isolated from canine pancreases by collagenase digestion and Euro-Ficoll purification. Following 12-h culture at 37 degrees C, islets were counted and dispensed into multiwell plate inserts. Islet function was assessed in each well immediately before and 24 h following a 10-min osmotic challenge with hypo- or hyperosmotic solutions of PBS (0, 75, 150, 300, 600, 1200, or 2300 mOsm/kg) at 22 degrees C. Canine islets reached their osmotic equilibrium within 10 min. Duplicate wells were used for each osmolality treatment for each of six donors (n = 12). No significant differences in basal or glucose-stimulated insulin secretion were found between wells prior to the osmotic challenge (3.35 +/- 0.45 and 20.98 +/- 3.36 microIU/IE/h, respectively). Following the osmotic challenge and 24-h in vitro tissue culture, a significant increase in basal secretion was observed for islets exposed to 0 and 75 mOsm/kg solutions and a significant decrease for islets exposed to 2300 mOsm/kg solution. Islets exposed to 0 and 2300 mOsm/kg solutions showed significant decreases in the stimulated insulin secretion when compared to controls. Solution osmolalities of 150-1200 mOsm/kg appear to be tolerated by canine islets with no significant deviations in insulin secretion. The corresponding tolerable volume range was 152.6 +/- 6.8% to 60 +/- 5.1% of the isotonic islet volume. The minimum critical volume was used in a theoretical analysis of the islet volumes that would result from equilibrium freezing in dimethyl sulfoxide (DMSO). The calculations show that 1.5 mol/l DMSO is sufficient to prevent damage to islets due to excessive shrinkage. Further refinement of cryoprotectant addition and dilution protocols, and cooling and warming protocols for canine islets, are now possible.
1-Methyl-3-isobutylxanthine↗
Water and cryoprotectant permeability characteristics of isolated human and canine pancreatic islets.
Cryopreservation allows accumulation of the necessary islet transplantable mass as well as adequate time for tissue typing and infectious disease screening. Cryopreservation protocols may be optimized by modeling the osmotically induced volume excursions that occur during the addition and removal of cryoprotective agents (CPAs). To that end, three transport parameters were measured at 22 degrees C in canine and human islets isolated by collagenase digestion and euroficoll purification: (i) the apparent hydraulic conductivity (Lp), (ii) the permeability coefficient of the CPA (Ps), and (iii) the associated reflection coefficient (sigma). The parameters were determined by volumetric analysis of islets upon abrupt exposure to 1, 2, and 3 M dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol (GLY), and propylene glycol (PG). The parameters were calculated using the Kedem-Katchalsky theory to describe islet volume excursion kinetics (assuming islets to be single equivalent osmotic units with the same volume and surface area of the actual islet) and a three-parameter curve fit was performed using the Marquardt-Levenberg method. It was determined that the permeability characteristics of pancreatic islets are species specific, and based upon the measured parameters, the highest Ps values for canine islets were observed following exposure to 2 M EG, and the highest Ps values for human islets were observed following exposure to 2 M PG. The permeability parameters were analyzed adjusting for islet radius using ANCOVA procedures to acquire least square means. For canine islets exposed to 2 M EG these values were determined to be 0.936 microm/min/atm, 2.47 microm/s, and 0.90 (for Lp, Ps, and phi, respectively) and for human islets exposed to 2 M PG the values were determined to be 1.56 microm/min/atm, 3.48 microm/s, and 0.85 (for Lp, Ps, and sigma, respectively). These parameters were used in a model to calculate osmotically induced islet volumetric response upon addition/dilution of the optimum CPAs, taking into consideration critical volume excursion limits at which irreversible damage occurs.
Animals↗
The effects of microencapsulation on pancreatic islet osmotically induced volumetric response.
Microencapsulation of pancreatic islets has been proposed as a means to prevent allograft rejection and to protect islets during cryopreservation. The aim of this study was to investigate: 1) the effects of the cryoprotectants (CPAs) dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on the volume of Ca2+ alginate microcapsules, and 2) the effects of microencapsulation on the volumetric response of human and canine pancreatic islets during CPA equilibration. Stock sodium alginate with a high mannuronic acid content (HM) or a high guluronic acid content (HG) was used to generate empty capsules (mean diameter 200 microm) with an electrostatic generator. The capsules were held in place by a holding pipette system and videotaped during the addition of 2 or 3 M CPA at 22 degrees C. Islets (isolated from human cadaveric donors and mongrel dogs and then cultured overnight at 37 degrees C) were encapsulated in alginate (HM), loaded into a microperfusion chamber, and the change in islet volume was videotaped after exposure to the same CPAs and concentrations. These were compared to the volume responses of nonencapsulated islets. Images were analyzed using a computerized image analysis system and the data were analyzed using ANOVA. HG microcapsules showed a significant (p < 0.05) increase in volume following exposure to EG but not to DMSO. HM microcapsule volume did not change significantly following exposure to either EG or DMSO and was therefore chosen as the substrate for islet encapsulation. Free, nonencapsulated canine and human islets responded to the osmotic challenge of the 2 M DMSO by shrinking to 70.00 +/- 1.04% (mean +/- SEM) and 70.11 +/- 1.05%, and in 2 M EG to 72.89 +/- 1.93% and 69.33 +/- 1.38%, respectively, of the isotonic volume before returning to the original cell volume. Exposure to 3 M DMSO or EG resulted in a further dehydration to 65.89 +/- 0.91% and 67.67 +/- 1.91% for canine and 62.22 +/- 0.66.% or 65.89 +/- 1.30% for human islets. Minimum volumes were reached within 30-40 s after exposure to the cryoprotectant. Encapsulated human islets reached 86.88 +/- 1.47% of their original volume in 2 M and 80.33 +/- 0.89% in 3 M DMSO, and 87.33 +/- 1.86% in 2 M and 82.80 +/- 1.57% in 3 M EG. This volume change was significantly less (p < 0.01) than that observed in corresponding free islets. Encapsulated canine islets reached 83.67 +/- 2.13% of their original volume in 2 M and 78.22 +/- 0.95% in 3 M DMSO, and 85.44 +/- 1.92% in 2 M and 78.11 +/- 2.01% in 3 M EG. As with human islets, this was significantly different than free islets (p < 0.01). These minimal volumes were reached within 30-50 s. These results demonstrate that there are cryoprotectant and alginate-specific interactions and that microencapsulation modulates the degree of osmotically induced shrinkage of islets. The development or modification of existing cryopreservation protocols to improve postcryopreservation recovery or function must account for these factors.
Animals↗
Measurement of human sperm intracellular water volume by electron spin resonance.
An electron spin resonance technique using the spin label tempone and the broadening agent potassium chromium oxalate was used to measure the water volume of human sperm. The toxicity of tempone (5 mmol/L) and potassium chromium oxalate (50 mmol/L) to sperm was measured over a time span of 120 minutes using computer-assisted semen analysis. Tempone had no effect on any computer-assisted semen analysis parameters, including motility. Potassium chromium oxalate reduced sperm motility by an average of 24% during the first 30 minutes of exposure. After selection by swim-up and correction for the presence of dead cells and cytoplasmic droplets, a water volume of 20.0 +/- 2.9 microns3 was obtained. This yields a total volume of 33.9 microns3 if a water compartment of 59% by volume is assumed. These results are consistent with other shape-independent techniques for measuring volume, but larger than the generally accepted optical and electronic particle counter sizes.
Blood Viscosity↗
Effects of passive motion and early vs. delayed ambulation on adhesion formation in rat uterine surgery.
The goal of our study was to assess the effects of unrestrained ambulation and of passive motion and delayed ambulation on the occurrence of adhesions after controlled uterine injuries, produced by conventional surgical techniques, in rats. A classic Pomeroy ligation was performed on the left uterine horn and a 1 cm x 3 mm rectangular flap hysterotomy on the right uterine horn in 48 rats, which were subsequently divided into four groups of 12 each. Group 1 served as controls and were allowed to ambulate without restriction following recovery from the anesthetic. Group 2 remained anesthetized for 12 hours, during which time each was passively moved from one lateral decubitus position to the opposite every 15 minutes. Group 3 were anesthetized for 12 hours and group 4 animals for 24 hours, following which unrestricted ambulation was allowed. All animals were killed 3 weeks after the operation and the left and right uterine horn adhesions were graded separately using a macroscopic system. The adhesion score of the left horn in group 2 animals (3.5) was significantly higher (P less than .025) than in the control animals (2.25). Group 3 animals had adhesion scores on the left (2.75), but did not differ significantly from controls (P greater than .05). Group 4 animals demonstrated significantly higher adhesion scores of the left uterine horn (3.3) than control animals. Group 2 animals, but not group 3 or group 4, had higher mean adhesion scores of the right uterine horn than control animals (P less than .005).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗