Stimulation of airway sensory nerves by cyclosporin A and FK506 in guinea-pig isolated bronchus

We have investigated the contractile property of cyclosporin A and FK506 in guinea-pig isolated bronchus. Cyclosporin A (10 microM) failed to significantly attenuate the excitatory non-adrenergic non-cholinergic (eNANC) and cholinergic contractile response (per cent methacholine Emax) induced by electrical field stimulation (EFS). In contrast, eNANC responses were significantly attenuated by both the neurokinin (NK)-1 and (NK)-2 receptor antagonists, N-acetyl-L-tryptophan 3,5-bis (trifluoromethyl)-benzyl and SR48968, respectively. Cyclosporin A and FK506 caused a concentration-dependent contraction in guinea-pig isolated bronchus, which was significantly attenuated by NK-1 and NK-2 receptor antagonists. The capsaicin receptor antagonist, capsazepine (10 microM) significantly reduced the contractile response to cyclosporin A and capsaicin, but not to FK506. The N-type calcium channel blocker, omega-Conotoxin (omegaCTX: 10 nM), significantly reduced the contractile response to FK506 and the eNANC response following EFS. In contrast, omega-CTX failed to significantly reduce the contractile potency to capsaicin or cyclosporin A. In bronchial preparations desensitized by repeated application of capsaicin (1 microM), the contractile responses to both cyclosporin A (100 microM) and FK506 (100 microM), were significantly reduced. In contrast, the contractile responses to substance P and neurokinin A (10 microM) were not altered. Furthermore, repeated application of cyclosporin A (100 microM) significantly inhibited the contractile response to capsaicin (1 microM). The findings from this study would indicate that cyclosporin A and FK506 mediate contraction of guinea-pig isolated bronchus secondary to the release of neuropeptides from airway sensory nerves. However, the release of sensory neuropeptides appears to be mediated via different mechanisms for cyclosporin A and FK506, the former by stimulation of the vanilloid receptor and the latter via opening of N-type calcium channels.     

Energy levels in resting and mitogen-stimulated human lymphocytes during treatment with FK506 or cyclosporin A in vitro

The effects of the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), on both the increase in intracellular free Ca2+ concentration ([Ca2+]i) and on the release of endogenous glutamate in rat hippocampal synaptosomes were studied. The inhibitory effect of CPA on the increase in [Ca2+]i stimulated with 4-aminopyridine was neutralized by the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The inhibitory effect of CPA was greater in synaptosomes from the CA1 subregion than in whole hippocampal synaptosomes. The inhibitory effects of both CPA and of the Ca2+ channel blockers, omega-conotoxin GVIA, omega-conotoxin MVIIC or omega-conotoxin GVIA plus omega-conotoxin MVIIC, were greater than those caused by the Ca2+ channel blockers. The release of endogenous glutamate was inhibited by 41% by CPA. The inhibition observed when CPA and omega-conotoxin GVIA or CPA and omega-conotoxin MVIIC were present was also greater than the inhibition by the Ca2+ channel blockers alone. The presence of both omega-conotoxin GVIA and omega-conotoxin MVIIC did not completely inhibit the release of glutamate, and CPA significantly enhanced this inhibition. The membrane potential and the accumulation of [3H]tetraphenylphosphonium of polarized or depolarized synaptosomes was not affected by CPA, suggesting that adenosine did not increase potassium conductances. The present results suggest that, in hippocampal glutamatergic nerve terminals, adenosine A1 receptor activation partly inhibits P/Q- and other non-identified types of Ca2+ channels.     

Comparative neurotoxic effects of root canal filling materials on rat sciatic nerve

The neurotoxic effects of the root canal filling materials--Endomethasone, N2 Universal, Traitement SPAD, Sealapex, and Calciobiotic Root Canal Sealer (CRCS)--were investigated on isolated rat sciatic nerves after local application. All of the canal filling materials reversibly inhibited the compound action potential (cAP) amplitudes. N2 Universal produced a 50% inhibition in 4.2 +/- 0.2 min. Traitement SPAD, Endomethasone, and CRCS produced the same inhibition in 6.4 +/- 0.3, 6.5 +/- 0.2, and 6.6 +/- 1.1 min, and Sealapex in 9.2 +/- 2.0 min. The inhibitory effect of Sealapex decreased fastest, and 43% recovery of cAP amplitude was observed in 60 to 70 min. The inhibitory effects of Endomethasone, CRCS, and N2 Universal were more pronounced, and 10 to 20% recovery in cAP amplitudes were observed in 2 h. The inhibitory effect of Traitement SPAD was more persistent with 4% recovery in 2.5 h.     

Peripheral nerve allograft preservation improves regeneration and decreases systemic cyclosporin A requirements

Peripheral nerve allografting is limited by the need for long-term systemic immunosuppression. The purpose of this study was to determine if nerve allograft preservation reduced the requirements for systemic Cyclosporin A (CsA) immunosuppression. One hundred twenty Lewis rats were randomized to one of seven experimental groups. Group 1 received a 2-cm Lewis posterior tibial nerve autograft. Groups 2-7 received 2-cm ACI posterior tibial nerve allografts. The allograft group was then further subdivided into three groups of two receiving daily subcutaneous injections of 0, 2.5, or 5.0 mg/kg of CsA for 12 weeks. Within each CsA dose, one group received a fresh while the other received a preserved allograft. Preserved grafts were stored in University of Wisconsin solution for 7 days at 5 degrees C prior to implantation. Animals from each group were sacrificed at 6, 12, and 20 weeks postoperatively. Evaluations included histomorphometry, electrophysiology, and serial walking track analysis. Histology revealed varying degrees of nerve regeneration in all groups at 6, 12, and 20 weeks. For a given CsA dose, the group receiving the preserved graft revealed evidence of better nerve regeneration by all histomorphometric parameters including fiber width and density, percentage neural tissue, and total fiber number. There was no statistical difference in walking track analysis between groups at 4 weeks. By 20 weeks, functional recovery statistically poorer than autograft was seen only in the fresh allograft groups receiving 0 or 2.5 mg/kg of CsA. Identical electrophysiologic findings were seen at 20 weeks. These results suggest that nerve graft preservation may decrease systemic immunosuppression requirements while improving functional recovery. As well, storage of nerve grafts is feasible and would facilitate elective surgery and less costly reconstructive repair.     

Nephrotoxicity studies of the immunosuppressants tacrolimus (FK506) and ascomycin in rat models

The nephrotoxic potential of ascomycin, the C21-ethyl analogue of FK506, was defined and ways explored to enhance its detection. After 14-day dosing in the Fischer-344 rat, FK506 and ascomycin reduced creatinine clearance by >50% at doses of 1 and 3 mg/kg, i.p., respectively. Ascomycin also had a 3-fold lower immunosuppressive potency in a popliteal lymph node hyperplasia assay, resulting in an equivalent therapeutic index consistent with a common mechanistic dependence on calcineurin inhibition. Renal impairment with different routes of administration was correlated with pharmacokinetics. Sensitivity of detection was not adequate with shorter dosing durations in rats with unilateral nephrectomy or in mice using a cytochrome P-450 inhibitor, SKF-525A. In 14-day studies, nephrotoxicity was not induced by continuous i.p. infusion of ascomycin at 10 mg/kg/day or daily oral administration (up to 50 mg/kg/day) in rats on a normal diet, nor by continuous i.v. infusion (up to 6 mg/kg/day) in rats on a low salt diet to enhance susceptibility. The lack of toxicity at high oral doses of FK506 or ascomycin, and the finding of non-linear oral pharmacokinetics of ascomycin show that this drug class has an oral absorption ceiling. The negative results with continuous infusion suggest that ascomycin nephrotoxicity is governed by peak drug levels. In addition to defining ways to meaningfully compare the nephrotoxic potential of FK506 derivatives, these results have implications for overall safety assessment and improved clinical use.     

Midgestational sciatic nerve transection in fetal sheep results in absent nerve regeneration and neurogenic muscle atrophy

In order to test whether fetal nerve healing and regeneration result in complete functional recovery, we transected the sciatic nerve at trunk level in 13 midgestational sheep fetuses. In 10 fetuses immediate microsurgical nerve coaptation was performed. The neonatal lambs were evaluated clinically, electrophysiologically, and histologically. On the transected side, the 10 surviving lambs showed a sensorimotor sciatic nerve paralysis and atrophy of the muscles innervated by the sciatic nerve. Somatosensory evoked potentials were weakly present in 5 animals and absent in 5 animals. Histologically, minimal signs of axonal regeneration, massive degeneration of the entire nerve, and a marked neurogenic muscle atrophy were found. These unexpected results differ from the findings after peripheral nerve transections in late gestational sheep fetuses and also from the classic wallerian degeneration-regeneration pattern that follows adult nerve injury. We speculate that the almost absent regenerative potential at midgestation is related to axotomy-induced neurotrophic factor deprivation during a developmental phase where the neurons are critically dependent on growth factor for survival.     

A comparison of tacrolimus (FK506) and cyclosporine for immunosuppression after cadaveric renal transplantation. FK506 Kidney Transplant Study Group

BACKGROUND: Tacrolimus (FK506), a macrolide molecule that potently inhibits the expression of interleukin 2 by T lymphocytes, represents a potential major advance in the management of rejection following solid-organ transplantation. This randomized, open-label study compared the efficacy and safety of tacrolimus-based versus cyclosporine-based immunosuppression in patients receiving cadaveric kidney transplants. METHODS: A total of 412 patients were randomized to tacrolimus (n=205) or cyclosporine (n=207) after cadaveric renal transplantation and were followed for 1 year for patient and graft survival and the incidence of acute rejection. RESULTS: One-year patient survival rates were 95.6% for tacrolimus and 96.6% for cyclosporine (P=0.576). Corresponding 1-year graft survival rates were 91.2% and 87.9% (P=0.289). There was a significant reduction in the incidence of biopsy-confirmed acute rejection in the tacrolimus group (30.7%) compared with the cyclosporine group (46.4%, P=0.001), which was confirmed by blinded review, and in the use of antilymphocyte therapy for rejection (10.7% and 25.1%, respectively; P<0.001). Impaired renal function, gastrointestinal disorders, and neurological complications were commonly reported in both treatment groups, but tremor and paresthesia were more frequent in the tacrolimus group. The incidence of posttransplant diabetes mellitus was 19.9% in the tacrolimus group and 4.0% in the cyclosporine group (P<0.001), and was reversible in some patients. CONCLUSIONS: Tacrolimus is more effective than cyclosporine in preventing acute rejection in cadaveric renal allograft recipients, and significantly reduces the use of antilymphocyte antibody preparations. Tacrolimus was associated with a higher incidence of neurologic events, which were rarely treatment limiting, and with posttransplant diabetes mellitus, which was reversible in some patients.     

Effects of riboflavin deficiency on the ultrastructure of rat sciatic nerve fibers

Ultrastructural studies indicate that riboflavin deficiency induced by either dietary restrictions alone or with the addition of the antagonist galactoflavin severely affects the structural integrity of myelin lamellae. The degenerative process induced by riboflavin deficiency is time dependent. Nonmyelinated nerve fibers are not affected ultrastructurally by the deficiency. Cellular organelles of both myelinated and nonmyelinated nerve fibers remain intact and presumably functional.     

Effects of cyclosporine and FK506 on in vitro high shear-induced platelet reactivity in rat and human non-anticoagulated blood

BACKGROUND: The immunosuppressants cyclosporine and FK506 have been used successfully in clinical transplantation, but both agents have various side effects. We have previously found that cyclosporine is prothrombotic and that FK506 is antithrombotic in an in vivo system. The aim of the present study was to assess the effects of these agents on platelet reactivity and coagulation using an in vitro shear-induced hemostatic platelet plug-forming instrument, the hemostatometer. METHODS: A purpose-built hemostatometer was constructed in our laboratory. The effects of cyclosporine and FK506 on platelet reactivity and coagulation were assessed under high shear stress using non-anticoagulated rat and human blood. RESULTS: FK506 significantly inhibited both platelet reactivity and coagulation. Cyclosporine also significantly inhibited coagulation, but a proaggregatory effect was observed at a final blood concentration of 0.05 mg/ml. CONCLUSIONS: The present in vitro results support our previous in vivo findings regarding the prothrombotic and antithrombotic effects of cyclosporine and FK506, respectively.     

Alteration in neurofilament axonal transport in the sciatic nerve of the diisopropyl phosphorofluoridate (DFP)-treated hen

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester that produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in hens 7-14 days after a single s.c. dose of 1.7 mg/kg. In this study, hens were treated with a single dose of DFP (1.7 mg/kg, s.c.) 24 hr after [35S]methionine injection into the sacrolumbar region of their spinal cord, and killed 3, 7, 14, or 27 days post-DFP treatment. The rates of transport of labeled high (NF-H), medium (NF-M), and low (NF-L) molecular weight neurofilaments, and tubulin were faster in DFP-treated birds than in controls after 3 days. Subsequently, the rate of transport of these proteins started falling, so that the peaks of labeled proteins in control and DFP-treated hens were overlapping after 7 days. At 14 days, the peaks of NF-H, NF-M, and NF-L in treated hens were distinctly behind the corresponding peaks in control hens. This was again followed by an increase in transport of NF-H and NF-L, but not of NF-M, so that the labeled NF-H and NF-L showed the same pattern in control and treated hens after 27 days. The transient decrease in NF-H and NF-L axonal transport rate, and recovery correlated in a temporal manner with the previously reported increase of Ca2+/calmodulin-dependent protein kinase-mediated phosphorylation of neurofilament proteins and inhibition of calpain activity in the sciatic nerve in OPIDN. Proteinase inhibition has been reported recently to result in enhanced phosphorylation of neurofilaments in some cells. The present study suggests that the enhanced phosphorylation of neurofilaments by DFP-increased Ca2+/calmodulin-dependent protein kinase activity may be contributing toward alteration in NF axonal transport and the development of OPIDN.     

Peripheral nerve glycoproteins and myelin fine structure during development of rat sciatic nerve

Developmental changes in relative amounts of peripheral nerve proteins and glycoproteins have been correlated with the degree of morphological myelination at various ages during the first 25 postnatal days in rat sciatic nerve. At birth there is virtually no major myelin glycoprotein (P0), but there is a protein which migrates to the same point on sodium dodecyl sulphate (SDS) polyacrylamide gels as the small myelin basic protein (P2). During the time myelin is being formed in the nerve, the P0 protein increases and the P2 protein appears to decrease in relative amount in the nerve. The accumulation of P0 protein in the nerve correlates extremely well with the degree of myelination in sciatic nerve. At 4-6 days postnatal, smooth membrane profiles are observed which are located within axons and in the inner Schwann cell cytoplasm. Such profiles are also observed to fuse with the axolemma-Schwann cell interface. The profiles may represent membrane material being added to or deleted from the axolemma or myelin during myelination.