Exogenous growth hormone attenuates cognitive deficits induced by intermittent hypoxia in rats.

Sleep disordered breathing (SDB), which is characterized by intermittent hypoxia (IH) during sleep, causes substantial cardiovascular and neurocognitive complications and has become a growing public health problem. SDB is associated with suppression of growth hormone (GH) secretion, the latter being integrally involved in the growth, development, and function of the CNS. Since GH treatment is able to attenuate neurocognitive deficits in a hypoxic-ischemic stroke model, GH, GH receptor (GHR) mRNA expression, and GH protein expression were assessed in rat hippocampus after exposures to chronic sustained hypoxia (CH, 10% O(2)) or IH (10% O(2) alternating with 21% O(2) every 90 s). In addition, the effect of GH treatment (50 µg/kg daily s.c. injection) on erythropoietin (EPO), vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1), and GLUT-1 mRNA expression and neurobehavioral function was assessed. CH significantly increased GH mRNA and protein expression, as well as insulin-like growth factor-1 (IGF-1). In contrast, IH only induced a moderate increase in GH mRNA and a slight elevation in GH protein at day 1, but no increases in IGF-1. CH, but not IH, up-regulated GHR mRNA in the hippocampus. IH induced marked neurocognitive deficits compared with CH or room air (RA). Furthermore, exogenous GH administration increased hippocampal mRNA expression of IGF-1, EPO, and VEGF, and not only reduced IH-induced hippocampal injury, but also attenuated IH-induced cognitive deficits. Thus, exogenous GH may provide a viable therapeutic intervention to protect IH-vulnerable brain regions from SDB-associated neuronal loss and associated neurocognitive dysfunction.

High fat/refined carbohydrate diet enhances the susceptibility to spatial learning deficits in rats exposed to intermittent hypoxia.

BACKGROUND: Intermittent hypoxia during sleep (IH), as occurs in sleep disordered breathing (SDB), induces spatial learning deficits associated with regulation of transcription factors associated with learning and memory in the hippocampal CA1 region in rats. high fat refined carbohydrate diet (HF/RC) can induce similar deficits and associated changes in signaling pathways under normoxic conditions. METHODS: Sprague-Dawley adult male rats were fed either with (HF/RC) or low fat/complex carbohydrate diet (LF/CC) starting at post-natal day 30 for 90 days, and were then exposed for 14 days during light phase (12 h/day) to either normoxia (RA) or IH (21% and 10% O2 alternations every 90 s). Place-training reference memory task deficits were assessed in the Morris water maze. Total and ser-133 phosphorylated CREB were assessed in different brain regions by Western blotting and immunostaining in rats exposed to normoxia or IH and to LF/CC or HF/RC. RESULTS: Substantial decreases in CREB phosphorylation occurred in CA1 but not in motor cortex following either IH, HF/RC, and HF/RC + IH. Place-training reference memory task deficits were observed in rats exposed to IH and to HF/RC, and to a much greater extent in rats exposed to HF/RC + IH. CONCLUSIONS: Nutritional factors alter recruitment of transcription factors, possibly via oxidative-related pathways, and modulate the vulnerability of the CA1 region of the hippocampus to the episodic hypoxia that characterizes SDB, thereby enhancing neurocognitive susceptibility in SDB patients.

Intermittent hypoxic exposure during light phase induces changes in cAMP response element binding protein activity in the rat CA1 hippocampal region: water maze performance correlates.

Intermittent hypoxia (IH) during sleep, a characteristic feature of sleep-disordered breathing (SDB) is associated with time-dependent apoptosis and spatial learning deficits in the adult rat. The mechanisms underlying such neurocognitive deficits remain unclear. Activation of the cAMP-response element binding protein (CREB) transcription factor mediates critical components of neuronal survival and memory consolidation in mammals. CREB phosphorylation and DNA binding, as well as the presence of apoptosis in the CA1 region of the hippocampus were examined in Sprague-Dawley male rats exposed to IH. Spatial reference task learning was assessed with the Morris water maze. IH induced significant decreases in Ser-133 phosphorylated CREB (pCREB) without changes in total CREB, starting as early as 1 h IH, peaking at 6 h-3 days, and returning toward normoxic levels by 14-30 days. Double-labeling immunohistochemistry for pCREB and Neu-N (a neuronal marker) confirmed these findings. The expression of cleaved caspase 3 (cC3) in the CA1, a marker of apoptosis, peaked at 3 days and returned to normoxic values at 14 days. Initial IH-induced impairments in spatial learning were followed by partial functional recovery starting at 14 days of IH exposure. We postulate that IH elicits time-dependent changes in CREB phosphorylation and nuclear binding that may account for decreased neuronal survival and spatial learning deficits in the adult rat. We suggest that CREB changes play an important role in the neurocognitive morbidity of SDB patients.

Bacterial components inhibit fibroblast proliferation in vitro.

Perigraft fluid from Staphylococcus epidermidis infected grafts in a mouse model significantly inhibits fibroblast proliferation (60-98% at 7 and 28 days), compared with perigraft fluid from sterile grafts. The fibroblast inhibitor was trypsin-heat resistant and dependent primarily upon the bacteria, not the host proinflammatory mediators or the vascular graft biomaterial. We tested the inhibitory properties of S. epidermidis strains RP62A (slime producer) and RP62NA (nonslime producer) and Staphylococcus aureus strain 502a, using an in vitro tritiated thymidine murine fibroblast (ATCC CCL-12) proliferation assay. Whole killed bacteria, disrupted bacteria (live and killed), bacterial supernatants, and purified cell wall products (peptidoglycan, teichoic acid, and lipoteichoic acid from disrupted bacteria) were studied. Significant fibroblast inhibition occurred for all three bacterial strains with disrupted bacteria (live or killed) and cell free bacteria derived supernatants. The fibroblast inhibitor from disrupted slime producing S. epidermidis was trypsin-heat resistant. The fibroblast inhibitor from disrupted S. aureus and supernatants for all three bacterial strains at 1 x 10(7) were trypsin-heat sensitive. Fibroblast inhibition was not dependent upon bacterial viability and not mediated by bacterial cell wall products. In conclusion, components of slime and nonslime producing S. epidermidis and S. aureus inhibit fibroblast proliferation.

Bacterial products primarily mediate fibroblast inhibition in biomaterial infection.

PURPOSE: The stimulation of fibroblast growth is essential for the normal healing and tissue integration of biomaterials. The local elevation of proinflammatory mediators in infected perigraft fluid (PGF) may inhibit this growth. We sought to determine whether infected PGF inhibited fibroblast growth, and, if so, whether this was primarily dependent on the biomaterial, bacteria, or host. METHODS: In vivo Dacron or expandable polytetra-fluoroethylene (ePTFE) grafts, sterile or colonized with slime-producing (RP-62A, viable or formalin-killed) or nonslime-producing (RP-62NA) Staphylococcus epidermidis (1 x 10(7) CFU/cm2), were implanted in Swiss Webster mice, and the PGF was harvested at 7 and 28 days. Antibodies to tumor necrosis factor alpha, interleukin 1 alpha, interferon gamma (7 micrograms/day), and indomethacin (50 micrograms/day) were administered by microinfusion pumps for 7 days and the PGF was harvested. Inhibition of the proinflammatory mediators was confirmed by enzyme-linked immunosorbant assay. The nontreated, heat-treated, or trypsin-digested in vivo PGF was incubated with an in vitro [3H]thymidine murine fibroblast (ATCC CCL-12) proliferation assay. RESULTS: Fibroblast inhibition was significant at 7 and 28 days with infected PGF incubation compared with sterile and was not dependent on bacterial slime production or viability. Dacron sterile PGF did not significantly inhibit fibroblasts compared with control, whereas sterile ePTFE stimulated (P < 0.05) fibroblasts. Treatment of the PGF with proinflammatory cytokines, heat, and trypsin failed to reverse fibroblast inhibition in the infected state. CONCLUSION: Biomaterial infection is associated with fibroblast inhibition that is dependent primarily on bacterial products and not the host or biomaterial. Conservative intervention strategies for graft infection need to address the problem of poor healing as well as bacterial clearance.

Staphylococcus epidermidis graft infection is associated with locally suppressed major histocompatibility complex class II and elevated MAC-1 expression.

OBJECTIVES: To determine the local cellular immune response in a series of human patients with Staphylococcus epidermidis prosthetic graft infection and to use a murine model to investigate the response in polytef (PTFE) and in a nonslime-producing S epidermidis variant. METHODS: Externally supported Dacron and PTFE grafts, either sterile or colonized with slime (RP-62A)- or nonslime (RP-62NA)-producing S epidermidis (10(7) colony forming units/cm2) were implanted in a dorsal subcutaneous pocket of Swiss Webster mice (Taconic, Germantown, NY). The grafts were harvested at 7, 10, 14, and 28 days with local bacterial and leukocyte counts obtained. Perigraft and blood monocyte major histocompatibility complex class II (MHC-II) (immune antigen) and membrane attack complex type 1 (MAC-1) (glycoprotein) expression were analyzed by flow cytometry in the murine model and in 3 patients representing 4 Dacron graft infections. RESULTS: The human infected Dacron perigraft monocytes revealed a suppressed MHC-II and elevated MAC-1 expression, and early correlation with the murine model was seen. No notable perigraft monocyte MHC-II suppression occurred in the infected PTFE graft. The reciprocal relationship in Dacron between monocyte MAC-1 and MHC-II expression was exaggerated with the lack of slime production. Bacterial clearance was variable. Supranormal expression was observed at 1 month in sterile Dacron but not in PTFE grafts. CONCLUSIONS: Staphylococcus epidermidis infection is associated with local cellular immune suppression in Dacron but not PTFE grafts. Slime-producing S epidermidis induced a lesser cytotoxic-phagocytic response than the nonslime variant. The reduced immunologic response to slime-producing S epidermidis may explain, in part, its indolent nature and resistance to eradication.

Prostaglandin E2 modulates monocyte MHC-II (Ia) suppression in biomaterial infection.

Staphylococcus epidermidis biomaterial infection is associated with local cellular immune suppression measured by a depressed monocyte (M phi) Ia expression. The purpose of this study was to define the effect of proinflammatory mediators on Ia expression and bacterial clearance in experimental biomaterial infection. A 1-cm-long Dacron tube graft, sterile or colonized with Staphylococcus epidermidis (1 x 10(7) cfu/ml2), was implanted in Swiss-Webster mice. Perigraft fluid was collected at 7, 10, 14, and 28 days and assayed by enzyme-linked immunoassays for tumor necrosis factor alpha (TNF alpha), interleukin (IL)-I alpha, IL-4, IL-10, and prostaglandin E2 (PGE2). Grafts were sonicated and plated for quantitative growth. In vivo effector inhibitions was accomplished with anti-TNF alpha, anti-IL-1 alpha antibodies (7 micrograms/24 hr), or indomethacin (50 micrograms/24 hr) via an Alzet 7-day microinfusion pump. M phi Ia expression was determined by flow cytometry. A significant elevation of TNF alpha, IL-1 alpha, and PGE2 was found during the first 10 days in the infected compared with sterile (P < or = 0.05) grafts and correlated with maximal Ia suppression. Neither IL-4 nor IL-10 was significantly different in the sterile or infected perigraft fluid. Indomethacin completely prevented M phi Ia suppression, while anti-IL-1 alpha only partially (94%) prevented M phi Ia suppression with a corresponding decrease in PGE2 production in infected grafts. Anti-TNF alpha increased PGE2 production by 189% and was associated with depressed M phi Ia expression. Indomethacin treatment improved mean graft-adherent bacterial clearance by 54% at 7 days and 75% at 28 days compared with control (not significant). Interleukin-1 alpha but not TNF alpha increases PGE2 production which modulates M phi Ia suppression. To improve treatment of biomaterial infections, local immunomodulation of PGE2 and IL-1 alpha is promising.

In vitro and in vivo effects of rifampin on Staphylococcus epidermidis graft infections.

Rifampin, bound in high concentrations to prosthetic grafts, has been proposed for the treatment of vascular graft infections. The optimum antibiotic concentration and duration of treatment for infected grafts is not known. This study compared the in vitro and in vivo efficacy of varying concentrations of rifampin against three different strains of slime producing Staphylococcus epidermidis (RP62A, KC2, and KB1) bound to the knitted Dacron at high and low concentrations at 10(4) and 10(7) CFU/cm2 of prosthetic. Time kill experiments were performed at 4, 18, and 42 hr, in which each Dacron bound bacterial strain was exposed in vitro to 4X, 64X, 100X, and 1,000X minimum inhibitory concentration (MIC) of rifampin. In vitro, the Dacron bound laboratory strain RP62A was implanted subcutaneously in the backs of male Swiss-Webster mice and exposed to 4X, 100X, and 1,000X the MIC of rifampin for similar time periods. In addition, systemic vancomycin (10 mg/kg) was assessed for synergy and prevention of rifampin resistance. In vitro, all concentrations of rifampin showed near total killing (< 1 log) of all bacterial strains at low initial concentrations (10(4) CFU/cm2) but not high (10(7) CFU/cm2) to 42 hr. Importantly, resistance was shown to develop in all three strains of S. epidermidis with high initial bacterial biofilm concentrations. In vivo, rifampin concentrations between 4X MIC and 100X MIC achieved a balance between optimal killing and prevention of resistance. Systemic vancomycin slightly improved bacterial clearance but did not alter the development of rifampin resistance at high local concentrations. Caution is advised with the use of antibiotic bonded grafts because resistance may develop even with the addition of systemic antibiotics.

Antibiotic efficacy against Staphylococcus epidermidis adherent to vascular grafts.

Antibiotic bonded grafts may improve the treatment results of vascular graft infections. The purpose of this study was to determine the antibiotic and antibiotic concentration needed to be effective against Staphylococcus epidermidis-infected vascular grafts. The efficacy of four antibiotics (minocycline, cefazolin, vancomycin, and rifampin) against S. epidermidis adherent to Dacron or Teflon vascular grafts was studied in vitro. Kill kinetic studies were performed with 18 and 42 hr of exposure of Dacron-adherent and Teflon-adherent S. epidermidis at 1, 4, 16, and 64 times the minimum inhibitory concentration (MIC) of each antibiotic. Antibiotic efficacy against graft-adherent S. epidermidis at 42 hr was best at concentrations 64x MIC for minocycline, cefazolin, and vancomycin and 4x MIC for rifampin. None of the antibiotics totally eradicated the graft-adherent bacteria. Antibiotics were equally effective for S. epidermidis adherent to Dacron and Teflon grafts. Antibiotic concentrations several times that predicted by the MIC were needed for all antibiotics to achieve significant killing of graft-adherent bacteria, with rifampin the most effective at the lowest concentration.

Immunosuppression augments growth of graft-adherent Staphylococcus epidermidis.

OBJECTIVE: To determine if systemic suppression of host defenses during graft implantation alters the initial adherence and subsequent growth of Staphylococcus epidermidis on vascular prostheses. DESIGN: Dacron grafts 1 cm2 were implanted in the back subcutaneous tissue of Swiss-Webster mice (n = 247), followed by topical inoculation with 2 x 10(7), 2 x 10(5), 2 x 10(3), or 2 x 10(1) colony-forming units of S epidermidis. Half of the mice were immunosuppressed with cyclophosphamide (150 mg/kg intraperitoneally), to achieve a consistent, significant decrease in the white blood cell count and major histocompatibility complex class II (Ia) expression. Control mice received an equal volume of saline solution. Graft bacterial biofilm concentrations were determined at 1 day for adherence and within 2 weeks for bacterial growth, by using sonication and quantitative agar culture. RESULTS: Immunosuppression did not significantly alter the initial adherence of bacteria to vascular grafts. Immunosuppressed animals that were inoculated with 2 x 10(7) and 2 x 10(5) colony-forming units of S epidermidis had significantly higher bacterial biofilm concentrations as compared with those in control animals. Graft infection persisted at 14 days in all animals, with and without immunosuppression. CONCLUSIONS: Suppression of immune function during graft implantation augmented growth of adherent bacteria. The effect of short-term perioperative immunosuppression on late-appearing S epidermidis graft infection needs further study.

The natural history of bacterial biofilm graft infection.

A mouse model was developed to study the natural history of vascular prosthetic graft infection due to Staphylococcus epidermidis. Graft infections were established in the back subcutaneous tissue of 46 mice by implantation of Dacron prostheses colonized in vitro with slime-producing S. epidermidis to form an adherent bacterial biofilm [1.7 x 10(7) colony forming units (CFU)/cm2 graft]. Control animals (n = 16) had implantation of sterile Dacron prostheses. None of the control animals developed a graft infection or graft-cutaneous sinus tract. All study animals developed a biofilm graft infection with typical anatomic (perigraft abscess), microbiologic (low bacterial concentration in surface biofilm), and immunologic (normal white blood count) characteristics. A graft-cutaneous sinus tract developed in a significantly higher number of mice with infected grafts by 8-10 weeks (9 of 21) compared to infected grafts explanted at 2 and 4-6 weeks (1 of 25, P < 0.01) and controls (0 of 16, P < 0.03). By 8-10 weeks, 2 animals had no signs of graft infection and the S. epidermidis study strain was not recoverable from 7 grafts. The natural history of bacterial biofilm vascular prostheses infection in the mouse model was similar to that in man, provoking a chronic inflammatory process curiously presenting as a perigraft abscess or graft-cutaneous sinus tract.