Interaction of formamidine pesticides with the presynaptic alpha(2)-adrenoceptor regulating.

The effects of the formamidine pesticides amitraz and chlordimeform on the alpha(2)-adrenergic receptor subtype that mediates the release of [(3)H]noradrenaline by synaptosomes from rat hypothalami were studied. We initially characterized the presynaptic autoreceptor on noradrenergic nerve endings using selective antagonists. Yohimbine (a nonselective alpha(2) antagonist) and BRL 44408 (selective for subtypes alpha(2A)/alpha(2D)) diminished the inhibitory effect of xylazine on K(+)-evoked release of [(3)H]noradrenaline; the K(B) values were 481 and 154 nM, respectively. In contrast, prazosin (a selective alpha(2B)/alpha(2C) antagonist) did not modify the inhibitory effect of xylazine. These results indicate that the release of noradrenaline by noradrenergic nerve endings in the rat hypothalamus is regulated by alpha(2D)-adrenoceptors, a species variation of the human alpha(2A) subtype. We then assessed the effects of the two pesticides on the K(+)-evoked release of [(3)H]noradrenaline. Amitraz reduced release in a dose-dependent manner; the effect observed at the maximal concentration tested (10 microM) was 13.0 +/- 2.0% and it was reversed by yohimbine. Amitraz also diminished the inhibitory effects of the alpha(2)-adrenergic agonists clonidine and xylazine. Chlordimeform displayed no effects, possibly because the true active compound of this insecticide is its demethylated metabolite. Based on these findings we conclude that the formamidine pesticides act as partial agonists of presynaptic alpha(2D)-adrenergic receptors in the rat hypothalamus. This interaction may be responsible for the in vivo alterations in catecholaminergic regulation of cyclic variations in gonadotropin-releasing hormone (GnRH) secretion, which can have grave functional repercussions on the reproductive system of mammals exposed to these xenobiotics.

Voltage-operated Ca(2+) channels involved in K(+)-evoked release of vasoactive intestinal polypeptide from the rat hypothalamus.

Rat brain hypothalami were exposed to various depolarizing stimuli and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) release was measured by means of a radioimmunoassay (RIA) procedure. Under conditions of noradrenergic blockade, exposure to high K(+) (40-100 mM) produced dose-dependent increases in the VIP-LI release in a Ca(2+)-dependent manner. Exposure to veratridine (3-100 microM) also induced concentration-dependent increases in VIP-LI release, an effect that was Ca(2+)-dependent and tetrodotoxin (TTX)-sensitive. Specific ligands for the L, N, and P/Q-type voltage-operated Ca(2+) channels (VOCCs) were used to determine which channel subtypes were involved in the K(+)-evoked VIP-LI release. The L-type VOCC ligand, nifedipine (10 microM), had no effect on release. In contrast, the N-type VOCC blocker, omega-conotoxin GVIA (omega-CgTx GVIA) (0.1-100 nM), markedly reduced the K(+)-evoked response, with maximal inhibition of approximately 60+/-8%. omega-Agatoxin IVA (omega-Aga IVA) (1-50 nM), which binds P-type and, at high doses, also Q-type VOCCs, produced dose-dependent inhibition of up to 25+/-3%, while the maximal inhibition observed with the non-selective VOCCs ligand, omega-conotoxin MVIIC (omega-CmTx MVIIC) (1 nM-3 microM), amounted to 85+/-8%. These findings indicate that N and P-type Ca(2+) channels play predominant roles in the high K(+)-evoked release of VIP-LI from the rat hypothalamus.

K(+)-Evoked [(3)H]D-aspartate release in rat spinal cord synaptosomes: modulation by neuropeptide Y and calcium channel antagonists.

This study was conducted to investigate mechanisms regulating the release of [(3)H]D-aspartate (or endogenous glutamate) in the rat spinal cord. Presynaptic modulation of glutamate release was studied in superfused synaptosomes depolarized with 20 mM KCl. Calcium-channel antagonists, omega-conotoxin GVIA (omega-CgTx GVIA; N-type), nifedipine (L-type), and omega-conotoxin MVIIC (omega-CmTx MVIIC; P/Q type), were used to characterize the voltage-operated Ca(2+) channels (VOCCs) involved in this release. Nifedipine had no significant effect on the K(+)-evoked release of [(3)H]D-aspartate, but the omega-conotoxins GVIA and MVIIC produced dose-dependent inhibitory effects that were additive. The most substantial reduction (54.30% +/- 4.40%) was seen with omega-CgTx GVIA, indicating that N-type channels play a major role in the release of glutamate in this tissue. We investigated the effects of neuropeptide Y (NPY), NPY(13-36), and [Leu(31)][Pro(34)]NPY on Ca(2+)-dependent, K(+)-evoked [(3)H]D-aspartate release. NPY and NPY(13-36) equipotently inhibited the release of glutamate in a concentration-dependent manner. The half-maximal response was observed at about 12 nM; maximal inhibition of 44.22% +/- 4.60% was achieved with 0.3 microM. The selective GABA(B) agonist (-)baclofen inhibited K(+)-evoked [(3)H]D-aspartate release from superfused spinal cord synaptosomes by 50.00% +/- 4.80% at 10 microM. When NPY(13-36) and (-)baclofen were used together at maximal doses, their release-inhibiting effects were not additive. In addition, neither of the agonists was able to enhance the inhibition produced by pretreating the synaptosomes with the selective inhibitor of N-type VOCCs omega-CgTx GVIA. These results are consistent with the hypothesis that presynaptic Y(2)-like and GABA(B) receptors regulate glutamate release by blocking Ca(2+) currents through N-type VOCCs. Characterization of the receptors that can inhibit the release of glutamate may provide useful information for treatment of conditions characterized by excessive glutamatergic transmission in the spinal cord.

Characterization of Ca(2+)-channels responsible for K(+)-evoked [(3)H]noradrenaline release from rat brain cortex synaptosomes and their response to amyotrophic lateral sclerosis IgGs.

The contribution of the different Ca(2+)-channel subtypes to the K(+)-evoked [(3)H]noradrenaline release from rat cerebral cortex synaptosomes has been investigated. In the same experimental model, it was also verified whether the calcium-mediated neurotransmitter release is influenced by IgGs purified from sera of seven patients affected by sporadic amyotrophic lateral sclerosis. Synaptosome treatment with 3.0 microM nifedipine or 2.0 microM calciseptine, which block L-type channels, slightly decreased [(3)H]noradrenaline release, the reduction being 7 and 13% of the control values, respectively. The blockade of N-type Ca(2+)-channels with omega-conotoxin-GVIA (0.001-1.0 microM) induced a concentration-dependent reduction of the neurotransmitter release, with maximum effect of 34%. omega-Agatoxin-IVA failed to significantly affect the studied release, which was instead markedly reduced by omega-conotoxin-MVIIC. After the blockade of N-type channels with maximal concentrations of omega-conotoxin-GVIA, 3.0 microM omega-conotoxin-MVIIC reduced the release by 58%. Synaptosome treatment with amyotrophic lateral sclerosis IgGs enhanced the K(+)-evoked [(3)H]noradrenaline release, which was mostly mediated by P/Q- and N-type Ca(2+)-channels. The increase induced by pathologic IgGs (0.2 mg/ml) ranged from 11 to 62% for the different patients, and it was concentration-dependent. The basal release was instead unaffected by IgG treatment. The results of the present study suggest that the K(+)-evoked [(3)H]noradrenaline release from brain cortex synaptosomes is mainly mediated by activation of P/Q- and N-type Ca(2+)-channels. Autoantibodies present in the sera of patients affected by sporadic amyotrophic lateral sclerosis may interact with these channels by producing an increased calcium influx, with consequent enhancement of the neurotransmitter release. Preliminary results of the present study have been published in abstract form (Martire et al., 1997, Pharmacol. Res. 35:9).

Effects of nitric oxide donors on basal and K+-evoked release of [3H]noradrenaline from rat cerebral cortex synaptosomes.

We investigated the effects of nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside on basal and K+-evoked release of [3H]noradrenaline from superfused synaptosomes from the rat cerebral cortex. Both substances produced concentration-dependent increases in the release of the labeled transmitter under basal and depolarized conditions. The effects of the donors on basal release were Ca2+-independent but were not inhibited by the carrier-uptake blocker, desipramine; the effects were abolished by hemoglobin (an NO scavenger). Thirty-five minutes after stimulation with sodium nitroprusside, the synaptosomes were still responsive to KCl stimulation, indicating that the donor's effects were not caused by damage to the synaptosome membrane. The cGMP analogue, 8-bromo-cGMP, had no effect on basal release, and the enhanced release produced by sodium nitroprusside was not inhibited by the specific inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, indicating that NO's effects on basal release of the neurotransmitter are guanylate cyclase-independent. Both of the NO donors had more marked effects on release of [3H]noradrenaline during K+-stimulated depolarization. The NO-mediated increase in this case was partially antagonized by 10 microM LH-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, and 8-Br-cGMP was also capable of producing concentration-dependent increases in the K+-stimulated release of the transmitter. These findings indicate that the effects of the NO donors on [3H]noradrenaline release during depolarization are partially mediated by the activation of guanylate cyclase.

The concept of the sagittal orbital-globe relationship in craniofacial surgery.

Euophthalmos, the normal relationship of the orbital rims to the eyes, is critical to planning and to surgical correction of craniofacial deformities. The four most easily localized anthropometric (soft tissue) landmarks for the sagittal orbital-globe relationship are orbitale superius (os), orbitale inferius (oi), orbitale laterale (ol), and nasion (n), all referenced to apex corneae (acor). The normal adult values for os, oi, ol, and n were extracted from the literature. Age-specific anthropometric landmarks were computed from age-specific Bolton cephalometric templates. A vernier caliper was used to measure preoperatively the surface orbital landmarks in patients with various syndromic and nonsyndromic craniosynostotic disorders. Preoperative measurements were compared with the derived normative data to determine the necessary sagittal orbital translocation for frontal advancement (n=19) and frontal-midfacial advancement (n=2). Postoperative orbital anthropometry documented the degree of normalization of the sagittal orbital-globe relationship. The problems with current instrumentation for orbital anthropometry are discussed.

Frontonasal suture expansion in the rabbit using titanium screws.

PURPOSE: This study investigated the ability to use titanium screws to provide stable skeletal points in the growing craniofacial region of the rabbit for suture expansion. These screws provided sites for direct application of external forces to alter growth and anatomic form. MATERIAL AND METHODS: Twenty-one rabbits (30 days old) were divided into three groups: control (n = 9), experimental (n = 9, and sham (n = 3). Four four-holed AO/ASIF commercially pure titanium craniofacial plates were contoured into an L-shape with a 90 degree angle at the midpoint. The plates were placed bilaterally in the frontal and nasal bone sites and secured with 2.0-mm diameter, 4-mm long, commercially pure titanium screws in both the sham and experimental groups. After 4 week of healing, a spring mechanism with a distraction force of 55 g was activated between ipsilateral plates and across the frontonasal sutures bilaterally in the experimental group for 5 weeks. No force was applied between the plates in the sham group. A preliminary evaluation of the bone-implant interface and the changes in the suture was done histologically. Morphologic changes were measured using cephalometric radiographs and direct anatomic measurements. RESULTS: The experimental group showed a significant increase in growth across the frontonasal suture in comparison with the sham group (P < .05). In addition, an increase in the length of the nasal and frontal bones in the expanded group was observed in comparison with the control and sham groups (P < .05). Histologically, a mixture of woven and lamellar bone was seen in the suture region and lamellar bone was seen in the screw-bone interface. CONCLUSION: This study indicates that titanium screws in the developing rabbit skull can provide stable sites for the direct application of external forces, producing secondary changes in skeletal morphology. This laboratory models provides a useful system for the further study of growth modification using such external mechanical forces.

Video registration virtual reality for nonlinkage stereotactic surgery.

We have combined three-dimensional (3D) computer-reconstructed neuroimages with a novel video registration technique for virtual reality-based, image-guided surgery of the brain and spine. This technique allows the surgeon to localize cerebral and spinal lesions by superimposing a 3D-reconstructed MR or CT scan on a live video image of the patient. Once the patient's scan has been segmented into the relevant components (e.g., tumor, edema, ventricles, arteries, brain and skin), the surgeon studies the 3D anatomy to determine the optimal surgical approach. The proposed intraoperative surgeon's perspective is displayed in the operating room at the time of surgery using a portable workstation. The patient is then brought to the operating room and positioned according to the planned approach. A video camera is trained on the patient from the proposed intraoperative surgeon's perspective. A video mixer merges the images from the video camera and the 3D computer reconstruction. This video mixer can vary the output intensity of the two input images between 100% of either and 50% of both. This visually superimposes the two images, not unlike a photographic double exposure. The patient's position and the 3D reconstruction are then adjusted until the images on the video mixer's output monitor are identical in terms of scale, position and rotation. This superimposition is facilitated by aligning various surface landmarks such as the external auditory canal, lateral canthus, and nasion. In some cases, such as with spinal tumors, capsules placed on the skin prior to scanning serve as fiducials.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer-assisted three-dimensional planning in craniofacial surgery.

Three-dimensional surface reconstruction from computed tomographic (CT) data has been used to plan craniofacial operations. Cephalometric and anthropometric databases were integrated with three-dimensional CT reconstructions to quantitate the skeletal deformity and to assist in the design of the surgical procedure. Interactive techniques were developed to simulate osteotomies and skeletal movements in three dimensions on the computer-generated surface images. The ocular globes were referenced to position the orbital segments; i.e., the osteotomized segments were transposed into normal anatomic relationship with respect to the eyes. The measurements from the computer graphic simulation were used intraoperatively to establish the correct position of the skeletal segments.

Enophthalmos following orbital transposition for craniofacial malformations.

This is a retrospective study of the frequency and factors that portend enophthalmos following orbital osteotomies and transposition for craniofacial malformations. Clinically obvious postoperative enophthalmos (POE) was noted in 23 (37.7 percent) of 61 patients undergoing such procedures. Postoperative enophthalmos was observed in 86 percent of Apert patients who had combined anteromedial orbital transposition and in 48 percent of patients with hypertelorbitism who had standard 360-degree osteotomies. In contrast, the incidence of postoperative enophthalmos was 21 percent following frontofacial (monobloc) or subcranial (Le Fort III) advancement. Postoperative enophthalmos also correlated with the occurrence of orbital fracture/fragmentation and with disruption of the periorbita. This study underscores the importance of establishing the correct relationship of the globe to the orbital rim (euophthalmos) while maintaining the spatial position of the eye, especially its anterior projection. Postoperative enophthalmos can be prevented by inserting bone grafts into orbital osteotomy gaps, correcting orbital volume/morphology following floor or wall outfracture/fragmentation, and preserving the periorbital supporting system.

Free and microvascular bone grafting in the irradiated dog mandible.

Microvascular and free rib grafts were placed in 4.5 cm defects in an edentate mandibular body defect 18 to 28 days after completion of 50 Gy of irradiation from a 60Co source. The animals were sacrificed from two to forty weeks postoperatively and evaluated clinically, radiographically, and histologically. There was a marked difference in the alveolar mucosal viability with the two grafts. Mucosal dehiscence was not observed over any of the microvascular grafts, but was present in seven-eighths of the free grafts. Union of the microvascular bone graft to the host bone occurred within six weeks. In contrast, after six weeks the free graft was sequestered in all the animals. An unexpected finding with both types of graft was the marked subperiosteal bone formation. This bone appeared to be derived from the host bed, stabilizing and bridging the defects bilaterally. The results suggest that radiated periosteum may play an important role in osteogenesis.

Fate of mineralized and demineralized osseous implants in cranial defects.

We have evaluated the fate of mineralized osseous implants placed into cranial defects in rats. By 2 weeks, 100% of the defects that had been filled with demineralized bone powder (DBP, 75-250 micrometer) showed bony repair as judged by histomorphometric analysis and incorporation of 45Ca. The DBP was not appreciably resorbed but rather was amalgamated within the new bone. Histomorphometric evaluation of osteo-genesis induced by equal masses of demineralized bone powders of various particle sizes (less than 75, 75-250, 250-450 micrometer) revealed that the smaller particles induced more bone per field than did the larger particles. In contrast, mineralized bone powder (BP) was completely resorbed by 3 weeks, without bony repair of the cranial defect. These specimens contained large multinucleated cells within 7 days and completely resorbed by 3 weeks. It is concluded that (a) demineralized bone powder predictably induces a osteogenic healing of cranial defects, (b) demineralized bone powder is not appreciably resorbed prior to bone induction, (c) the extent of bone induction is a function of the surface area of the demineralized bone implant, and (d) mineralized bone powder undergoes obligatory resorption.