Prenatal blockade of estradiol synthesis impairs respiratory and metabolic responses to hypoxia in newborn and adult rats.

We tested the hypothesis that estradiol modifies respiratory control in pregnant rats and participates in the development of respiratory chemoreflexes in fetuses. Pregnant rats (n = 12) received daily subcutaneous injections of vehicle (Veh, n = 6) or 4-androsten-4-ol-3,17-dione acetate (ATD; inhibitor of estradiol synthesis; n = 6; 5 mg/day in vehicle) from gestational day 16 (G16) to delivery. Baseline ventilation (whole body plethysmography) and metabolic rate [oxygen consumption (Vo(2))] were determined at G14 and G20, in pups [on postnatal day 3 (P3) and P20] and in adult rats (on P70) born to Veh- or ATD-treated mothers. Hypoxic chemoreflex was assessed in P3 rats by acute exposure to 60% O(2) and in P20 or P70 rats by moderate hypoxia (12% O(2), 30 min). ATD treatment reduced circulating estradiol in pregnant dams at G20 without producing changes in the circulating level of estradiol precursors (testosterone and androstenedione). ATD-treated dams showed impaired respiratory adjustment to late gestation. Pups born to ATD mothers had higher resting Vo(2) (+23% at P3, +21% at P20), respiratory frequency (+15% at P3, +12% at P20), and minute ventilation (+11% at P3, +18% at P20) than pups from Veh mothers. Respiratory decrease during acute hyperoxic exposure at P3 was -9.7% in Veh (P < 0.05 vs. room air) and only -2.6% (P = not significant) in ATD pups. In P20 ATD rats, hypoxic ventilatory response was attenuated compared with Veh. In P20 and P70 rats, the drop of Vo(2) in hypoxia (-31% in P70, P < 0.0001) was not observed in ATD rats. We conclude that estradiol secreted during late gestation is necessary for respiratory adjustment to pregnancy and is required for adequate development of respiratory and metabolic control in the offspring.

Spinal cord reconstruction using NeuroGel implants and functional recovery after chronic injury.

There is currently a lack of effective ways to achieve functional tissue repair of the chronically injured spinal cord. We investigated the potential of using NeuroGel, a biocompatible polymer hydrogel, to induce a reconstruction of the rat spinal cord after chronic compression-produced injury. NeuroGel was inserted 3 months after a severe injury into the post-traumatic lesion cavity. Rats were placed in an enriched environment and the functional deficits were measured using the BBB rating scale. A significant improvement in the mean BBB scores was observed. Rats without enriched environment and severely injured rats with an enriched environment alone showed no improvement; however, 7 months after reconstructive surgery using NeuroGel, a reparative neural tissue had formed within the polymer gel that included myelinated axons and dendro-dendritic contacts. NeuroGel implantation into a chronic spinal cord injury therefore resulted in tissue reconstruction and functional improvement, suggesting that such an approach may have therapeutic value in the repair of focal lesions in humans.

Reconstruction of the transected cat spinal cord following NeuroGel implantation: axonal tracing, immunohistochemical and ultrastructural studies.

This study examined the ability of NeuroGel, a biocompatible porous poly [N-(2-hydroxypropyl) methacrylamide] hydrogel, to establish a permissive environment across a 3 mm gap in the cat spinal cord in order to promote tissue reconstitution and axonal regeneration across the lesion. Animals with NeuroGel implants were compared to transection-only controls and observed for 21 months. The hydrogel formed a stable bridge between the cord segments. Six months after reconstructive surgery, it was densely infiltrated by a reparative tissue composed of glial cells, capillary vessels and axonal fibres. Axonal labelling and double immunostaining for neurofilaments and myelin basic protein, showed that descending supraspinal axons of the ventral funiculus and afferent fibres of the dorsal column regenerated across the reconstructed lesion. Fifteen months after reconstructive surgery, axons had grown, at least, 12 mm into the distal cord tissue, and in the rostral cord there was labelling of neurons of the intermediate gray matter. Electron microscopy showed that after 9 months, most of the regenerating axons were myelinated, principally by Schwann cells. Newly formed neurons presumably from precursor cells of the ependyma and/or migrating neurons were observed within the reparative tissue after 21 months. Results indicate that functional deficit, as assessed by treadmill training, and morphological changes following double transection of the spinal cord can be modified by the implantation of NeuroGel. This technology offers the potential to promote the formation of a neural tissue equivalent via a reparative neohistogenesis process, that facilitates and supports regenerative growth of axons.

In vivo magnetic resonance imaging and relaxometry study of a porous hydrogel implanted in the trapezius muscle of rabbits.

In vivo magnetic resonance imaging (MRI) and relaxometry were performed to assess noninvasively the tissue reaction and the biological integration of hydrogels made of poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) after implantation in the trapezius muscle of rabbits. The benefits of incorporating RGD peptide sequences in the polymer backbone were also investigated. The histological status of each implant was probed by the trend of their transversal relaxation times, T(2), while their biocompatibility was evaluated by analyzing the host tissue response through the evolution of the relaxation times of the adjacent muscle tissue. MR results showed the good acceptability of both hydrogels by the host tissue. The transversal relaxation curves of each implant exhibited two distinct phases as a function of implantation time: (1) a monoexponential phase, dominated by the influx of fluids inside the implants; and (2) a biexponential phase related to the infiltration of cells and the granulation tissue formation within the porous structure of each polymer. These MR findings were correlated with the results of conventional histological analyses. The present study demonstrates the effectiveness of MR methods in noninvasively monitoring the biocompatibility and histological status of implanted porous biomaterials.

Noradrenoceptor antagonism with idazoxan improves L-dopa-induced dyskinesias in MPTP monkeys.

Treatment of Parkinson's disease with L-dopa is plagued in a majority of patients by dyskinesias. Noradrenaline/dopamine interactions are proposed on behavioral, biochemical, physiological and anatomical grounds. The aim of the study was to test the potential antidyskinetic effect of the alpha2-adrenoceptor antagonist, idazoxan, in a primate model of Parkinson's disease. Six female cynomolgus monkeys previously rendered parkinsonian by the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and presenting an unchanged syndrome for several months were used. All responded readily to L-dopa but had developed dyskinesias which were manifested with each dose. In the first part of the study, seven doses of idazoxan (ranging from 0.25 mg/kg to 10 mg/kg, p.o.) were administered together with the vehicle or in combination with a fixed dose of L-dopa/benserazide (100/25 mg, p.o.). In the second part of the study, a fixed dose of idazoxan (7.5 mg/kg) was administered daily for 10 days and L-dopa was added to idazoxan on days 1, 4, 7 and 10. Vehicle (empty capsule) was used as control. Idazoxan, by itself (ranging from 5 mg/kg to 10 mg/kg), increased locomotor activity and improved the disability score with virtually no dyskinesias in three animals. In combination with L-dopa, idazoxan did not impair the antiparkinsonian response but significantly reduced dyskinesias in all six animals up to 65% at doses of 7.5 mg/kg and 10 mg/kg and delayed their onset, so that the "ON" state without dyskinesias was prolonged. The antidyskinetic effect of idazoxan was maintained when repeatedly administered for 10 days. On day 10, the locomotor response to L-dopa was significantly potentiated by chronic administration of idazoxan. Our results indicate that idazoxan has some antiparkinsonian effect of its own and may constitute a useful adjunct to L-dopa as it can reduce dyskinesias without impairing the relief of symptoms, this effect being maintained over time in this model.

Effect of the selective D1 antagonists SCH 23390 and NNC 01-0112 on the delay, duration, and improvement of behavioral responses to dopaminergic agents in MPTP-treated monkeys.

We assessed the antiparkinsonian response in MPTP-treated monkeys after acute or repeated treatment with oral L-Dopa, subcutaneous administration of L-Dopa methyl ester (LDME) or apomorphine, alone and in combination with D1 antagonists SCH 23390 (SCH) or NNC 01-0112 (NNC). When given alone, the L-Dopa effect occurred within the first hour after treatment. Coadministration of SCH or NNC with L-Dopa significantly delayed the onset of action. The response duration remained unchanged, as did the extent of the antiparkinsonian effect, after SCH, whereas the former became shorter at the higher doses of NNC tested. Bypass of the gastrointestinal tract using parenteral injections of LDME and apomorphine allowed the rapid turning "on" of the animals. Both D1 antagonists administered with LDME delayed the onset and shortened the duration of the therapeutic effect as the dose increased. Pretreatment with SCH failed to block the antiparkinsonian effect induced by apomorphine, but reduced the response duration markedly in a dose-related fashion. Repeated treatment of one monkey with SCH combined with the same dopaminergic drugs produced results similar to those obtained after acute treatment in four animals. The results obtained with parenteral administration of LDME and apomorphine most probably involve pharmacodynamic actions resulting in increased threshold of response. The delay observed with L-Dopa suggests pharmacokinetic interference possibly mediated via dopamine receptors located at the level of the gut.

D1 receptor blockade improves L-dopa-induced dyskinesia but worsens parkinsonism in MPTP monkeys.

OBJECTIVE: To determine whether dopamine (DA) D1 or DA D2 receptors are associated predominantly with the antiparkinsonian versus the dyskinetic effect of levodopa. METHODS: The authors used four L-dopa-primed, dyskinetic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-exposed monkeys to test whether acute and selective blockade of the DA D1 receptor subtype, using SCH 23390 and NNC 01-112, could reduce L-dopa-induced dyskinesias without altering the relief of symptoms. Blockade of DA receptors using sulpiride (D2) and clozapine (D1-D2-like) was studied for comparison. RESULTS: With the notable exception of the lowest dose of clozapine tested, coadministration of DA D1 or D2 antidopaminergic agents with L-dopa reduced the L-dopa-induced dyskinesias but also caused a return of parkinsonian disability. Prolonged latencies from intake of a single oral dose of L-dopa to turning "on," decreased duration of the "on" state, and a complete failure to induce benefit was also observed. CONCLUSION: Low-dose clozapine could be an effective adjunct to reduce L-dopa-induced dyskinesias without altering the relief of parkinsonian symptoms. Interactions with many neurotransmitter systems may explain the better pharmacologic profile of clozapine, including DA D4 (rather than D1), serotonin, acetylcholine, and noradrenaline. Neither dyskinesias nor antiparkinsonian effects can be ascribed solely to the D2 or D1 receptor. Thus, some cooperation between the two receptors appears necessary for these behavioral effects.

Distribution of 7-alkyl-2'-deoxyguanosine adduct levels in human lung.

Human lung tissue is frequently studied as a target organ for DNA damage from carcinogen-DNA adducts. In order to assess the distribution of carcinogen-DNA adducts in human lung, we measured 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-methyl-dGp), 7-ethyl-2'-deoxyguanosine-3'-monophosphate (7-ethyl-dGp) and 4-hydroxy-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in different lobes. The first two result from exposure to N-nitrosamines, including tobacco-specific nitrosamines, and the latter only from tobacco-specific nitrosamines. Using a chemically-specific 32P-postlabeling assay for 7-alkyl-2'-deoxyguanosines, adducts were measured in eight separate lung segments of ten autopsy donors. 7-Methyl-dGp levels were detected in all eighty samples (range from 0.3 to 11.5 adducts/10(7) dGp; mean 2.5 +/- 2.3 adducts/10(7) dGp). 7-Ethyl-dGp were detected in all but five of the samples (range from <0.1 to 7.1 adducts/10(7) dGp; mean 1.6 +/- 1.7 adducts/10(7) dGp). 7-Methyl-dGp levels were approximately 1.5-fold higher than 7-ethyl-dGp levels, and they were positively correlated with each other in most individuals. There was no consistent pattern of adduct distribution in the different lobar segments. Most individuals, especially those with the lowest levels, had similar levels among the lobes, while those with the highest levels had a widely variable pattern ranging as much as ten-fold. Moreover 7-methyl-dGp and 7-ethyl-dGp levels in all people showed a highly significant inter-individual variation (P = 0.0001). The levels of 7-alkyl-2'-deoxyguanosine among individuals could not be explained by differences in tobacco exposure (measured by serum cotinine), onset of death, gender, age, race, blood ethanol, or ventilation and perfusion variability. In an effort to corroborate 7-alkyl-2'-deoxyguanosine adducts variability among lobes or individuals, we sought to determine a correlation with HPB-releasing DNA adducts as an independent marker of tobacco exposure. However, this tobacco- specific carcinogen-DNA adduct could not be detected in four individuals tested (detection limit: 0.3 adducts per 10(7) dGp). Based upon the lack of 7-alkyl-2'-deoxyguanosine discernible adduct patterns, no conclusions could be drawn regarding a potential relationship to lobar cancer incidence. The results indicate that in studies of carcinogen-DNA adducts, such as 7-alkyl-dGp in human lungs, for most individuals a random lung sample would be representative of other parts of the lungs. Some individuals however might be misclassified due to highly variable 7-alkyl-dGp levels.

A single-cell study of the axonal projections arising from the posterior intralaminar thalamic nuclei in the rat.

Thalamostriatal projections arising from the posterior intralaminar nuclei (P1; the parafascicular nucleus and the adjacent caudalmost part of the posterior thalamic group) were studied in rats by tracing the axons of small pools of neurons labelled anterogradely with biocytin. Thirteen P1 cells were also stained by juxta cellular application of the tracer. Relay cells of P1 nuclei have a morphology that differs radically from the classical descriptions of the bushy cells which represent the main neuronal type of the sensory thalamic relay nuclei. P1 cells have ovoid or polygonal somata of approximately 20-25 microm, from which emerge four or five thick, long and poorly branched dendrites bearing spines and filamentous appendages; their dendritic domains extend for up to 1.5 mm. Before leaving the nucleus 20% of axons give off collaterals that ramify locally. All axons course through the thalamic reticular nucleus, where they also distribute collaterals, and arborize massively in the striatum and sparsely in the cerebral cortex. At the striatal level four or five collaterals leave the main axon and terminate in patches scattered dorsoventrally within a rostrocaudally oriented slab. As revealed by calbindin D-28k immunohistochemistry, only the matrix compartment receives terminations from P1 axons. The cortical branch form small terminal puffs centred upon layer VI of the motor cortex. Before entering the striatum some axons of the parafascicular nucleus give rise to descending collaterals that arborize in the entopeduncular nucleus, in the subthalamic nucleus and in the vicinity of the red nucleus. Other axons arising from the caudal part of the posterior group send descending branches only to the entopeduncular nucleus. These findings show that P1 cells belong to a distinct category of thalamic relay neurons which, beside their massive projection to the striatum, also distribute collaterals to other components of the basal ganglia. Moreover, these results provide the first direct evidence that virtually all P1 cells project to both striatum and cerebral cortex. Finally, it is proposed on the basis of morphological, histochemical and hodological criteria that the caudal part of the posterior thalamic group in the rat is homologous to the suprageniculate-limitans nuclei of cats and primates.