Optimising perioperative care for hip and knee arthroplasty in South Africa: a Delphi consensus study.

BACKGROUND: A structured approach to perioperative patient management based on an enhanced recovery pathway protocol facilitates early recovery and reduces morbidity in high income countries. However, in low- and middle-income countries (LMICs), the feasibility of implementing enhanced recovery pathways and its influence on patient outcomes is scarcely investigated. To inform similar practice in LMICs for total hip and knee arthroplasty, it is necessary to identify potential factors for inclusion in such a programme, appropriate for LMICs. METHODS: Applying a Delphi method, 33 stakeholders (13 arthroplasty surgeons, 12 anaesthetists and 8 physiotherapists) from 10 state hospitals representing 4 South African provinces identified and prioritised i) risk factors associated with poor outcomes, ii) perioperative interventions to improve outcomes and iii) patient and clinical outcomes necessary to benchmark practice for patients scheduled for primary elective unilateral total hip and knee arthroplasty. RESULTS: Thirty of the thirty-three stakeholders completed the 3 months Delphi study. The first round yielded i) 36 suggestions to preoperative risk factors, ii) 14 (preoperative), 18 (intraoperative) and 23 (postoperative) suggestions to best practices for perioperative interventions to improve outcomes and iii) 25 suggestions to important postsurgical outcomes. These items were prioritised by the group in the consecutive rounds and consensus was reached for the top ten priorities for each category. CONCLUSION: The consensus derived risk factors, perioperative interventions and important outcomes will inform the development of a structured, perioperative multidisciplinary enhanced patient care protocol for total hip and knee arthroplasty. It is anticipated that this study will provide the construct necessary for developing pragmatic enhanced care pathways aimed at improving patient outcomes after arthroplasty in LMICs.

Exploratory behaviour and novel predator recognition: behavioural correlations across contexts.

It was hypothesized that the exploratory behaviour of an individual measured in a novel environment could predict its behaviour in response to a novel predator. This study examined novel predator recognition in the western mosquitofish Gambusia affinis, a species with individual differences in risk-taking, activity and exploration in novel environments. Prey responded with characteristic shoaling and avoidance in response to native predators, but did not show characteristic antipredator behaviour towards novel predators. Furthermore, G. affinis exhibited individual-level behavioural correlations across contexts but only when prey were tested with native predators. This could be the result of native predatory selection on behavioural correlations in the prey species.

Single cycle to failure in torsion of three standard and five locking plate constructs.

OBJECTIVES: The biomechanical properties of standard plates and recently designed locking plates were compared in torsion. We hypothesized that titanium (Ti) constructs would have the greatest deformation, and String of Pearls (SOP) constructs the greatest strength and stiffness. METHODS: Dynamic compression plates (DCP), stainless steel (SS) limited contact (LC)-DCP, Ti LC-DCP, locking compression plate (LCP), 10 mm and 11 mm Advanced Locking Plate System (ALPS) 10 and 11, SOP and Fixin plates were applied to a validated bone model simulating a bridging osteosynthesis. Yield torque (strength), yield angle (deformation) and stiffness were compared using one-way ANOVA with post hoc Tukey (p <0.05). RESULTS: The ALPS 11 constructs had significantly greater elastic deformation than all constructs except for the ALPS 10. There were not any differences in strength observed except for the ALPS 10 constructs, which was less than that for the SOP, LCP, DCP and ALPS 11 constructs. No differences in construct torsional stiffness were observed with the SS LC-DCP, DCP, LCP and SOP constructs; however all had greater stiffness than all remaining constructs. The ALPS 10 construct had lower stiffness than all constructs. CLINICAL SIGNIFICANCE: Modulus of elasticity of Ti explains the higher deformation and lower stiffness of these systems, with similar results for the Fixin due to its lower section modulus compared to all other plates. The SOP and standard constructs had surprisingly similar biomechanical properties in torsion. The rationale for selecting these implants for fracture repair likely needs to be based upon their differing biomechanical properties inherent to the diverse implant systems.

Single cycle to failure in bending of three standard and five locking plates and plate constructs.

OBJECTIVE: To evaluate the biomechanical properties of standard and locking plates in bending. We hypothesised that titanium (Ti) constructs would have the greatest deformation and that String of Pearl (SOP) constructs would have the greatest strength and stiffness, and would behave differently compared to plates alone. METHODS: Dynamic compression plates (DCP), stainless steel (SS) limited contact (LC)-DCP®, Ti LC-DCP, locking compression plates (LCP), 10 mm and 11 mm advanced locking plate system (ALPS 10 / 11), SOP and Fixin plates were evaluated individually and as constructs applied to a validated bone model simulating a bridging osteosynthesis. Bending stiffness and strength were compared using one-way ANOVA with post hoc Tukey, and unpaired t-test (p <0.05). RESULTS: The SOP plates had significantly greater stiffness than all other plates; Ti LC-DCP, ALPS 10 and Fixin plates had significantly lower stiffness than all other plates. The SOP constructs had the highest mean bending stiffness, and strength that was significantly different from only the Ti LC-DCP, ALPS 10 and Fixin constructs. The ALPS 10 constructs had the lowest mean bending stiffness, and strength that was significantly different from only ALPS 11 and SOP constructs. Comparison of bending structural stiffness of plates versus constructs showed a significant difference in all plate pairs except for the DCP and ALPS 10. CLINICAL RELEVANCE: Due to differing plate construct properties inherent to these diverse implant systems, identical approaches to fracture management and plate application cannot be applied.

Quality improvement in acute stroke: the New York State Stroke Center Designation Project.

BACKGROUND: Many hospitals lack the infrastructure required to treat patients with acute stroke. The Brain Attack Coalition (BAC) published guidelines for the establishment of primary stroke centers. OBJECTIVE: To determine if stroke center designation and selective triage of acute stroke patients improve quality of care. METHODS: Baseline chart abstraction was performed on all stroke patients admitted to 32 hospitals serving Brooklyn and Queens, NY, from March to May 2002. Hospitals were invited to meet BAC guideline-based criteria. Adherence was verified by on-site visits. After designation, acute stroke patients were selectively triaged. Remeasurement data were collected from August to October 2003. RESULTS: The authors abstracted 1,598 charts at baseline and 1,442 charts at remeasurement. From baseline to remeasurement, median times decreased for door to physician contact (25 vs 15 minutes, p = 0.001), CT performance for potential tissue plasminogen activator (t-PA) candidates (68 vs 32 minutes, p < 0.001), and t-PA administration (109 vs 98 minutes (p = NS). IV t-PA utilization increased from 2.4 to 5.2% (p < 0.005), select t-PA protocol violations decreased from 11.1 to 7.9% (p = NS), and the stroke unit admission rate increased from 16 to 39% (p < 0.001). In stroke centers (n = 14) vs nondesignated hospitals (n = 18), there were shorter median times from door to physician contact (10 vs 25 minutes, p < 0.001), CT performance for potential t-PA candidates (31 vs 40 minutes, p = NS), and t-PA administration (95 vs 115 minutes, p < 0.05). Stroke centers, compared with nondesignated centers, admitted acute stroke patients to stroke units more often (55.9 vs 10.9%, p < 0.001). CONCLUSIONS: Stroke center designation and selective triage of acute stroke patients improved the quality of care, including access to timely thrombolytic therapy and stroke units.

rhBMP-2 injected in a calcium phosphate paste (alpha-BSM) accelerates healing in the rabbit ulnar osteotomy model.

This study evaluated the ability of recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered in an injectable calcium phosphate carrier (alpha-BSM) to accelerate healing in a rabbit ulna osteotomy model compared to untreated surgical controls. Healing was assessed by radiography, histology and biomechanics. Bilateral mid-ulnar osteotomies were created in 16 skeletally mature rabbits. One limb in each animal was injected with either 0.1 mg rhBMP-2/alpha-BSM (BMP) (N=8) or buffer/alpha-BSM (BSM) (N=8). Contralateral osteotomies served as untreated surgical controls (SXCT). Gamma scintigraphy showed 75%, 45% and 5% of the initial 125I-rhBMP-2 dose was retained at the osteotomy site at 3 h, 1 week and 3 weeks. The biological activity of rhBMP-2 (alkaline phosphatase activity from bioassay) extracted from alpha-BSM incubated in vitro up to 30 days at 37 degrees C was unchanged. Radiographs demonstrated complete bridging of the BMP limbs at 4 weeks whereas none of the BSM or SXCT limbs were bridged. Post-mortem peripheral quantitative computed tomography determined mineralized callus area was 62% greater in BMP limbs compared to SXCT limbs. Torsional stiffness and strength were 63% and 103% greater in BMP limbs compared to SXCT limbs. There was no difference in torsional properties between BSM and SXCT limbs. Failure occurred outside the osteotomy in four out of seven of the BMP limbs. All BSM and SXCT limbs failed through the osteotomy. Histology showed bony bridging of the osteotomy and no residual carrier in the BMP limbs. BSM and SXCT groups showed less mature calluses composed of primarily fibrocartilaginous tissue and immature bone in the osteotomy gap. These data indicate rhBMP-2 delivered in alpha-BSM accelerated healing in a rabbit ulna osteotomy model compared to BSM and SXCT groups.

Recombinant human bone morphogenetic protein-2 enhances osteotomy healing in glucocorticoid-treated rabbits.

The objectives of this study were to evaluate the effect of chronic prednisolone treatment on osteotomy healing in rabbits and to determine whether recombinant human bone morphogenetic protein-2 (rhBMP-2) would enhance healing in the presence of chronic glucocorticoid therapy. Forty-nine skeletally mature, male rabbits were injected with either prednisolone (n = 26; 0.35 mg/kg per day, three times a week) or saline (n = 23). After a 6-week pretreatment period, bilateral ulnar osteotomies were created surgically. One osteotomy was treated with rhBMP-2 (0.2 mg/ml of rhBMP-2, 40 microg of rhBMP-2 total) delivered on an absorbable collage sponge (ACS), whereas the contralateral osteotomy remained untreated. Prednisolone or saline treatment was continued until the rabbits were killed either 6 weeks or 8 weeks after creation of the osteotomy. Osteotomy healing was evaluated by radiography, peripheral quantitative computed tomography (pQCT), torsional biomechanics, and undecalcified histology. Because we observed similar responses to both prednisolone and rhBMP-2/ACS treatment in the 6-week and 8-week cohorts, the results from these time points were combined. Serum osteocalcin and vertebral trabecular bone density were lower in the prednisolone-treated rabbits. Prednisolone treatment dramatically inhibited osteotomy healing. In the untreated ulnas, callus area and torsional strength were 25% and 55% less, respectively, in the prednisolone-treated rabbits than in the saline group (p < 0.001 for both). rhBMP-2/ACS enhanced healing in both the prednisolone- and the saline-treated groups, although the effect was larger in the prednisolone-treated rabbits. In the prednisolone-treated rabbits, callus area and torsional strength were 40% and 165% greater (p < 0.001 for both), respectively, in osteotomies treated with rhBMP-2/ACS compared with the contralateral, untreated osteotomies. Histological evaluation confirmed that osteotomy healing was inhibited by prednisolone and accelerated by rhBMP-2/ACS. In summary, a single application of rhBMP-2/ACS counteracted the inhibition of osteotomy healing caused by prednisolone exposure. These results suggest that rhBMP-2/ACS may be a useful treatment for enhancing fracture healing in patients who are undergoing chronic glucocorticoid therapy.

Recombinant human bone morphogenetic protein-2 accelerates healing in a rabbit ulnar osteotomy model.

BACKGROUND: Approximately 5% to 20% of fractures have delayed or impaired healing. Therefore, it is desirable to develop new therapies to enhance fracture-healing that can be used in conjunction with traditional treatment methods. The purpose of this study was to evaluate the ability of a single application of recombinant human bone morphogenetic protein-2 to accelerate fracture-healing in a rabbit ulnar osteotomy that heals spontaneously. METHODS: Bilateral mid-ulnar osteotomies (approximately 0.5 to 1.0 mm wide) were created in seventy-two skeletally mature male rabbits. The limbs were assigned to one of three groups: those treated with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2, those treated with an absorbable collagen sponge containing buffer, and those left untreated. In the first two groups, an 8 20-mm strip of absorbable collagen sponge containing either 40 g of recombinant human bone morphogenetic protein-2 or buffer only was wrapped around the osteotomy site. The rabbits were killed at two, three, four, or six weeks after surgery. In addition, twenty-four age-matched rabbits were used to provide data on the properties of intact limbs. The retention of recombinant human bone morphogenetic protein-2 at the osteotomy site was determined with scintigraphic imaging of (125)I-labeled recombinant human bone morphogenetic protein-2. After the rabbits were killed, the limbs were scanned with peripheral quantitative computed tomography to assess the area and mineral content of the mineralized callus. The limbs were then tested to failure in torsion, and undecalcified specimens were evaluated histologically. RESULTS: Gamma scintigraphy of (125)I-recombinant human bone morphogenetic protein-2 showed that 73% +/- 6% (mean and standard deviation) of the administered dose was initially retained at the fracture site. Approximately 37% +/- 10% of the initial dose remained at the site one week after surgery, and 8% +/- 7% remained after two weeks. The mineralized callus area was similar in all groups at two weeks, but it was 20% to 60% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 than in either the ulnae treated with buffer or the untreated ulnae at three, four, and six weeks (p < 0.05). Biomechanical properties were similar in all groups at two weeks, but they were at least 80% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 at three and four weeks than in either the ulnae treated with buffer (p < 0.005) or the untreated ulnae (p < 0.01). By four weeks, the biomechanical properties of the ulnae treated with recombinant human bone morphogenetic protein-2 were equivalent to those of the intact ulnae, whereas the biomechanical properties of both the ulnae treated with buffer and the untreated ulnae had reached only approximately 45% of those of the intact ulnae. At six weeks, the biomechanical properties were similar in all groups and were equivalent to those of the intact ulnae. The callus geometry and biomechanical properties of the ulnae treated with buffer were equivalent to those of the untreated ulnae at all time-points. CONCLUSIONS AND CLINICAL RELEVANCE: These findings indicate that treatment with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2 enhances healing of a long-bone osteotomy that heals spontaneously. Specifically, osteotomies treated with recombinant human bone morphogenetic protein-2 healed 33% faster than osteotomies left untreated. The results of this study provide a rationale for testing the ability of recombinant human bone morphogenetic protein-2 to accelerate healing in patients with fractures requiring open surgical management.

Interactions between estrogen, tamoxifen, octylphenol, and two polychlorinated biphenyls in murine splenocytes.

Prior exposure of cultured murine splenocytes to 17beta-estradiol (E) protects them from the membrane disrupting effects of the xenoestrogen 4-tert-octylphenol (OP). Using splenocytes isolated from male Balb/c mice, we tested whether (a) the xenoestrogen, 2', 3', 4', 5'-tetrachloro-4-biphenylol (PCB-OH), or the polychlorinated biphenyl, 3, 3', 4, 4'-tetrachlorobiphenyl (PCB 77), which displays both estrogenic and anti-estrogenic actions, would compromise the membrane integrity of the cells and (b) E or tamoxifen (TX), another ligand for the E receptor, would protect the membranes of cells exposed to the agents. We also examined possible interactions between OP, PCB-OH, and PCB 77 on the cells. Splenocytes were cultured for 24 hr. Concentrations of OP (10(-5)-10(-9) M), PCB-OH (10(-6)-10(-16) M), or PCB 77 (10(-8)-10(-12) M) significantly compromised the membrane integrity of the cultured splenocytes in a dose response manner. Concentrations of E as high as 10(-5) M or TX as high as 10(-7) M were without effect. Incubation of splenocytes in medium containing E or TX at 10(-7) M for 2 hr prior to the subsequent addition of either OP, PCB-OH or PCB 77 (final concentrations of 10(-7), 10(-7), or 10(-8) M, respectively) blocked the membrane disrupting effects. Incubation of splenocytes in medium containing 10(-7) M E starting 2 hr after the addition of OP or PCB 77 or incubation of splenocytes in medium containing 10(-7) M TX starting 2 hr after the addition of OP or PCB-OH did not block the damaging effects of OP, PCB 77, or PCB-OH on the cell membranes. No interactions were observed when various combinations of OP, PCB-OH, or PCB 77 were used. These data suggest that: (a) TX acts like E in this system, (b) a prior response of splenocytes to E or TX can protect them from the potential cytotoxic effects of OP, PCB-OH, or PCB 77; and, (c) OP, PCB-OH, and PCB 77 were not additive in their actions.

Toxic effects of octylphenol on cultured rat spermatogenic cells and Sertoli cells.

Alkylphenols, including the estrogenic 4-tert-octylphenol (OP), are environmental pollutants. Because administration of OP to adult male rats impairs spermatogenesis and OP has been shown to be toxic to aquatic animals and to mammalian splenocytes in vitro, we studied whether OP exerts direct toxic effects on cultured spermatogenic cells and Sertoli cells isolated from male rats. Cell viability was assessed with a Live/Dead Eukolight viability/cytotoxicity kit. Culture of mixed spermatogenic cells from adult rats with 10(-8) M OP or Sertoli cells from 19- to 21-day-old rats with 10(-12) M OP, but not with 0.08% EtOH (vehicle) or 10(-6) M 17beta-estradiol (E2) or dexamethasone (DEX), significantly decreased the percentage of viable cells after 24 h of treatment. None of the treatments significantly altered total cell number. Flow cytometric analyses of spermatogenic cells revealed that exposure to 10(-4) or 10(-6) M OP yielded abnormal relative ploidy classes. Four hours of treatment with 10(-6) M OP, but not with 10(-6) M E2 or DEX, caused significant chromatin condensation in Sertoli cells as observed with acridine orange staining. The decreased percentage of viable cells after 24 h of exposure to 10(-6) M OP remained when Sertoli cells were cultured in Ca2+-free medium. Sertoli cells contained nuclei of reduced size and labeled 3'-OH DNA ends as detected by microscopic analyses when the cells had been incubated for 24 h with 10(-6) M OP but not with vehicle or 10(-6) M E2 or DEX. The results demonstrate that OP, but not E2 or DEX, is directly toxic to cultured rat spermatogenic cells and Sertoli cells and suggest that this toxic effect in Sertoli cells is exerted through Ca2+-independent apoptosis.

Disruption of rat estrous cyclicity by the environmental estrogen 4-tert-octylphenol.

4-tert-Octylphenol (OP) is a prevalent environmental pollutant which binds to estrogen receptors and exerts estrogenic actions in vitro. The effects of OP in vivo on mammalian female reproduction are not known. We investigated whether (i) exposure of neonatal rats to OP interfered with the onset of vaginal opening or their ability to have regular estrous cycles as adults and (ii) exposure of adult rats to OP interfered with estrous cyclicity and ovulation. Injection of 1 mg OP in corn oil sc on the day after birth did not affect the day of vaginal opening. However, 9 of 11 OP-treated rats were in persistent vaginal estrus when examined at three months after birth compared with 0 of 9 corn oil-injected controls, which cycled regularly. Ten of eleven neonatal rats injected with 1.7 mg of the estrogenic pesticide methoxychlor also were in persistent estrus at 3 months after birth, and all 10 neonatal rats injected with 1 mg of 2,4,5-trichlorophenol, which is apparently nonestrogenic, cycled regularly. Injection of 20 or 40 mg OP in corn oil vehicle sc three times weekly into previously untreated adult cyclic rats caused persistent estrus in 2 of 6 and 16 of 21 rats, respectively. Injections were continued for three more weeks in 5 of the 16 rats rendered persistent estrus by the 40 mg OP treatment. These rats remained in persistent estrus for the additional 3-week period. The other 11 persistent estrous rats in the 40 mg treatment group started to cycle regularly within 5-7 days after the last injection. Unlike pentobarbital, injection of OP into cyclic rats during the afternoon of proestrus did not block ovulation. These results provide strong evidence that OP acts like estrogen in vivo in both neonatal and adult female rats to exert effects that block reproductive cyclicity.

Chronic administration of the environmental pollutant 4-tert-octylphenol to adult male rats interferes with the secretion of luteinizing hormone, follicle-stimulating hormone, prolactin, and testosterone.

4-Tert-octylphenol (OP) is a prevalent environmental pollutant that has been shown to exert both toxic and estrogenic effects on mammalian cells. The effects of OP on the reproductive system of adult male vertebrates are virtually unknown. In the present study, we investigated the effects of chronic exposure to OP on reproductive hormone secretion in the adult male rat and compared the results qualitatively with those observed in other male rats treated chronically with estrogen. We injected corn oil vehicle or OP (20 or 80 mg) or estradiol valerate (EV; 0.8 or 8 microg) in oil s.c. into 2-mo-old male rats thrice weekly for either 1 or 2 mo. The 80-mg dosage of OP and one or both dosages of EV had the following effects: decreased anterior pituitary gland (APG) and serum LH and FSH concentrations; increased APG and serum prolactin (PRL) concentrations; increased APG/body weight ratios; decreased serum testosterone concentrations; decreased hematocrit; and decreased food consumption and body weight gain. To evaluate the response of the hypothalamus-APG to gonadal removal, we orchidectomized some of the rats after the end of treatment and decapitated them 3 wk later. In orchidectomized controls, serum LH and FSH concentrations rose markedly and serum PRL concentrations decreased. Similar changes were seen in orchidectomized rats treated previously with 20 or 80 mg OP. Moreover, there were no differences in mean serum LH, FSH, or PRL concentrations between controls and rats treated previously with either dosage of OP at 3 wk after orchidectomy. The results demonstrate that chronic administration of OP to adult male rats can adversely affect the secretion of reproductive hormones and strongly suggest that OP exerts these effects by acting like an estrogen. The opposite changes in LH, FSH, and PRL secretion observed after cessation of treatment with OP and orchidectomy suggest that chronic treatment with OP under the conditions of the present study did not result in any significant permanent deleterious effects on gonadotrophs or lactotrophs or the hypothalamic neurons controlling the secretion of the gonadotropins or PRL.

Chronic administration of 4-tert-octylphenol to adult male rats causes shrinkage of the testes and male accessory sex organs, disrupts spermatogenesis, and increases the incidence of sperm deformities.

The environmental toxicant 4-tert-octylphenol (OP) has been shown to exert estrogenic effects on mammalian cells in culture. Recent findings from our laboratories demonstrate clearly that OP administration disrupts reproductive hormone secretion in the adult male rat, quite likely as a result of estrogenic action. In the present study, we investigated the impact of these or other OP-induced changes on male reproductive tissues. Adult male rats were injected with OP (20 or 80 mg) or estradiol valerate (EV; 0.8 or 8 microg) s.c. in oil three times a week for either 1 or 2 mo. We found that an 80-mg dosage of OP for 2 mo or an 8-microg dosage of EV for 1 or 2 mo greatly reduced sperm numbers and adversely influenced the sizes, weights, and histological structures of the testes, epididymides, ventral prostate glands, seminal vesicles, and coagulating glands. The 80-mg dosage of OP for 1 mo reduced epididymal tubule size to a lesser extent than after 2 mo of treatment. Otherwise, treatment with 80 mg OP for 1 mo, 20 mg OP for 1 or 2 mo, or 0.8 microg EV for 1 mo had little or no effect on the histology of the tissues we examined. Additional evaluation of sperm morphology revealed marked increases in the proportions of head and tail abnormalities from animals that had received 80 mg of OP or 8 microg of EV for 1 mo and 20 mg of OP for 2 mo. The head abnormalities consisted mainly of pin heads, detached heads, and the absence of hooks, while tail abnormalities included mainly broken, coiled, and bent tails. Our results clearly demonstrate that OP can severely reduce the size and/or function of all of the male gametogenic and accessory reproductive organs studied. Moreover, the similarity of these cell and tissue changes between rats treated with OP and those treated with EV further suggests that OP may exert its action in an estrogenic-like manner.

Estrogen can protect splenocytes from the toxic effects of the environmental pollutant 4-tert-octylphenol.

Four-tert-octylphenol (OP), an environmental pollutant, exerts apoptotic effects on cultured mouse splenocytes. Although OP binds to estrogen receptors, these apoptotic effects are not exerted by 17 beta-estradiol (E). It remained possible that OP might bind to estrogen receptors and subsequently exert apoptotic effects not exerted by E after it binds to the same receptors. It also remained possible that E-primed splenocytes might respond to OP differently than splenocytes not exposed to E. Thus, we investigated OP and E interactions on the viability of mouse splenocytes in culture. The total number of splenocytes (cells stained and not stained with trypan blue) was not altered or altered slightly after incubation with any agent for 24 h. Incubation of splenocytes in medium containing 5 x 10(-5) or 5 x 10(-7) M OP decreased the percentage of viable cells by only approx 47% and 25%, respectively. The addition of 0.8 x 10(-5) to 0.8 x 10(-9) M E to cultures was without effect or decreased the percentage of viable cells by only approx 5%. The addition of these concentrations of E simultaneously with or at 2 h after the addition of 5 x 10(-5) M or 5 x 10(-7) M OP to cultures did not interfere with the OP-induced decreases in cell viability. By contrast, incubation of splenocytes in medium containing E for 2 h prior to the subsequent addition of either dose of OP blocked the OP-induced decreases in cell viability in a dose-response manner. There was a marked reduction in the percentage of viable cells (70%) when splenocytes were incubated with 0.5 x 10(-5) M dexamethasone. The addition of 0.8 x 10(-5) M E at 2 h prior to the addition of dexamethasone did not prevent the decreased cell viability. Incubation of cells in medium with 0.8 x 10(-5) M testosterone caused a small decrease in splenocyte viability similar to that observed with E. However, unlike E, the addition of testosterone at 2 h prior to the addition of 5 x 10(-5) M OP did not prevent the OP-induced decrease in cell viability. These data suggest the presence of estrogen receptors in some splenocytes. They also suggest that if OP binds to these estrogen receptors or other receptors in the absence or initial presence of E, the resulting effect is toxic to the cells. By contrast, exposure of splenocytes to E prior to their exposure to OP can prevent the toxicity of OP.

Toxic effects of octylphenol on cultured rat and murine splenocytes.

Alkylphenol polyethoxylates and alkylphenols, such as 4-tertoctylphenol (OP), are environmental contaminants. Because these compounds are toxic to aquatic animals, we studied the effects of OP on splenocytes removed from male Fischer 344 rats or male Balb/c mice and cultured in vitro. Cell viability was assessed by trypan blue exclusion after 5 or 27 hr of culture. Culture with 0.08% ETOH (vehicle) or any dose of OP did not alter total cell number or the percentage of viable cells after 5 hr. Culture of cells with two different alkylphenol polyethoxylates for 5 hr resulted in the loss of all cells. The percentages of viable rat or mouse cells after 27 hr of culture were decreased significantly by 10(-12) M OP or greater concentrations. The actions of OP, dexamethasone (DEX), and 17 beta-estradiol on rat splenocytes were compared. Dexamethasone was more toxic than OP after 24 hr of culture; 17 beta-estradiol was not toxic. Dexamethasone and OP, but not 17 beta-estradiol, caused significant nuclear condensation after 3 hr of culture (acridine orange staining) or 4 hr of culture (propidium iodide staining). The toxicity of 10(-6) M OP, but not that of 10(-6) M DEX, was eliminated when mouse splenocytes were cultured in Ca2+ -free medium. Significantly more mouse splenocytes containing free 3'-OH DNA ends were detected by activated cell sorter analyses when the cells had been incubated for 4 hr with 10(-4) or 10(-6) M OP or 10(-6) M DEX. The results of these studies demonstrate that OP is toxic to cultured rat and mouse splenocytes and suggest that this toxic effect is exerted, at least partially, through Ca2+-dependent apoptosis.

Adenohypophysial allografts releasing prolactin decrease prolactin mRNA concentration in the host hamster's adenohypophysis in situ.

The inhibitory effects of pituitary allografts on the prolactin (PRL)-secretory system are presumed to be consequences of the unabated release of PRL by the allografts. In the present studies we used pituitary allografts in the Golden Syrian hamster to address the following questions: (a) Do allografts of adult adenohypophysial tissue which elevate serum PRL levels decrease the concentration of PRL mRNA in the host's adenohypophysis? (b) Is this effect shared by allografts of neonatal hypophysial tissue or neonatal muscle tissue which do not elevate serum PRL levels? (c) Do any of these types of allograft alter growth hormone mRNA in the host's adenohypophysis? Prolactin mRNA concentration, but not growth hormone mRNA concentration, was decreased in the adenohypophyses in situ in the hosts bearing adult adenohypophysial allografts in which serum PRL levels were elevated. In contrast, serum PRL in hosts with neonatal hypophysial or muscle allografts were not elevated and PRL mRNA levels in the adenohypophysis in situ were not decreased when compared to the levels measured in hamsters with sham transplants. Prolactin mRNA levels in hosts with neonatal muscle allografts were not different from levels in hosts with neonatal hypophysial allografts but were increased when compared to the levels measured in hamsters with sham transplants. There were no differences in PRL concentration in the adenohypophyses in situ between any of the groups. Also, PRL concentrations in neonatal hypophysial allografts were similar to those in adult adenohypophysial allografts. To our knowledge these observations are the first demonstrating that short-loop feed-back of PRL includes a decrease in PRL mRNA concentration. The observations also support the working hypothesis that PRL and not another pituitary factor exerts the negative feedback.

Changes in percentages of adenohypophysial gonadotrophs associated with the sex-specific, selective increase in serum follicle-stimulating hormone concentration in the juvenile female hamster.

In normal hamsters, we investigated whether the sex-specific, selective increase in serum FSH concentration in the juvenile female was associated with sex-specific changes in the percentages of adenohypophysial gonadotrophs. Serum LH concentrations did not rise between Day 4 and Day 19 in either sex and did not differ significantly between the sexes on Days 4, 7, 12, 14, and 19 after birth. Serum FSH concentrations were about 2-fold higher on Days 7, 12, and 14 than on Days 4 or 19 in males. In females, serum FSH rose markedly between Days 4 and 7, declined slightly by Day 12, rose to peak levels by Day 14, and declined slightly by Day 19 to levels not different from those seen on Day 7. Body weights rose between Days 4 and 19 and were similar in both sexes. There were no sex differences in pituitary gland weights, which rose between Days 4 and 12 and did not increase significantly further by Day 19. On Day 0, the percentages of immunoreactive LH and FSH cells were about 6 and 1%, respectively, in both sexes. These percentages increased progressively between Days 0 and 7 and between Days 7 and 14. On Day 7, but not on Day 14, the percentages of LH and FSH cells were greater in females than in males. There were more LH than FSH cells in males on Days 0, 7 and 14, and in females on Day 0 but not on Day 7 or 14. Matching of 10 FSH cells per gland with LH cells in serial sections of each of 30 glands showed FSH immunoreactivity to occur only in cells staining for LH. In hypophysectomized-gonadectomized adult hamster hosts with allografts of neonatal pituitary glands beneath the renal capsule, we investigated whether these sex-specific changes in the percentage of cells might be predetermined by the time of birth or dependent on sex differences in the internal environment existing in the postnatal hamster. Groups consisted of male donors-male hosts, male donors-female hosts, female donors-female hosts, and female donors-male hosts. The percentages of LH cells in allografts in all four groups increased from Days 0 to 7 and from Days 7 to 14. Percentages of LH cells on Day 14 in all four groups were not different from those in age-matched male or female adenohypophyses in situ. In contrast, the mean percentages of FSH cells were low (about 1-3%) on Days 0, 7, and 14 in all four groups. In other males hosts, administration of a low dose of LHRH for 7 days did not alter the percentage of LH cells in male allografts but increased the percentage of FSH cells to approach that observed in age-matched male adenohypophyses in situ. Administration of a larger dose of LHRH for 7 days to other male hosts with male allografts increased the percentages of LH and FSH cells to percentages not different from those in age-matched female adenohypophyses in situ. Matching of 10 FSH cells/allograft with LH cells in serial sections of each of 58 allografts showed FSH immunoreactivity to occur only in cells staining for LH. The results of experiments conducted on normal hamsters demonstrate that more marked increases in the percentages of adenohypophysial LH cells and FSH cells occur in females than in males in association with the onset of the selective increase in serum FSH levels in females. The results of experiments employing allografts suggest that the greater increase in LH and FSH cells in females is due to sex differences in the internal environment existing in the postnatal hamster, which can be accounted for by differences in LHRH secretion, rather than to inherent differences between female and male adenohypophyses at the time of birth. We conclude that the greater increases in gonadotrophs observed in female hamster pups on Day 7 after birth and the accompanying sex-specific, selective elevation in serum FSH concentration are probably due to sex differences in LHRH secretion during the juvenile period.

Neuropeptide Y and luteinizing hormone releasing hormone synergize to stimulate the development of cellular follicle-stimulating hormone in the hamster adenohypophysis.

Luteinizing hormone releasing hormone (LHRH) stimulates the development of cellular FSH immunoreactivity in the perinatal hamster adenohypophysis. Because neuropeptide Y (NPY) can act directly on rat adenohypophysial cells to stimulate FSH and LH release and potentiate the stimulatory effect of LHRH on FSH and LH release, we investigated the effects of NPY alone and in combination with a low, ineffective dose of LHRH on inducing cellular FSH immunoreactivity in the neonatal hamster adenohypophysis. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized-ovariectomized adult hamster hosts with a catheter implanted in the external jugular vein. After treatment, hosts were decapitated and graft tissue was stained for FSH and LH immunoreactivity. The mean percentage of adenohypophysial cells that stained for FSH was low (2.8%) in grafts in hosts infused continuously with heparinized saline vehicle for 7 days. In other hosts, peptides were pulsed through the catheter every 12 h for 7 days. The mean percentage of FSH cells also was low after pulsing 6 ng LHRH or 2 micrograms NPY but increased substantially when the two peptides were pulsed simultaneously. No differences in the mean percentage of LH cells existed between any of the groups. The results demonstrate that NPY and LHRH can synergize to induce cellular FSH immunoreactivity in the neonatal female hamster.

Induction of cellular follicle-stimulating hormone in the hamster adenohypophysis requires intermittent stimulation by luteinizing hormone releasing hormone.

We investigated the effectiveness of continuous vs intermittent LHRH stimulation of the neonatal female anterior pituitary gland on inducing cellular FSH immunoreactivity in the Golden Syrian hamster. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized-ovariectomized adult hosts with a catheter implanted in the external jugular vein. In experiment 1, vehicle or LHRH (6 ng/h) was infused continuously or LHRH was pulsed at 1 h (6 ng) or 12 h (72 ng) intervals through the catheters for 8 days. Hamsters were decapitated for collection of trunk blood shortly after the end of treatment, and grafts were prepared for immunocytochemical staining for LH and FSH. Anterior pituitary glands removed from neonatal (day 1) and day 9 female pups also were stained for LH and FSH. The mean percentage of adenohypophysial cells staining for LH increased from 11% in neonatal pups to mean percentages (24-28%) that were similar in day 9 pups and in all groups with grafts. The mean percentage of adenohypophysial cells staining for FSH increased from 1% in neonatal pups to percentages (16-21%) that were similar in day 9 pups and in grafts in hosts administered 6 or 72 ng LHRH pulses. By contrast, the mean percentage of FSH cells did not increase in grafts in hosts administered vehicle or LHRH by continuous infusion. Serum LH concentration was low in hosts given vehicle or LHRH by continuous infusion but elevated in hosts given 72 ng LHRH pulses and in all but one host given 6 ng LHRH pulses.(ABSTRACT TRUNCATED AT 250 WORDS)