Human cadavers preserved using Thiel's method for the teaching of fibreoptically-guided intubation of the trachea: a laboratory investigation.

We assessed the suitability of human cadavers preserved using Thiel's method for teaching flexible fibreoptic tracheal intubation. Thirty-one anaesthetists unacquainted with this technique received didactic teaching followed by handling of the fibrescope on the Oxford teaching box. They then carried out fibreoptic intubations in two cadavers to establish a baseline sample of their intubation skills. Thereafter, we randomly assigned the trainees to two groups to practice fibreoptic intubation either on two distinct cadavers or on two airway manikins. After 7 days we re-assessed procedural skills using the same cadavers as at baseline. Intubation time was the primary outcome and secondary outcomes included the incidence of failed intubations. We also evaluated trainee satisfaction. The mean (SD) intubation time decreased from a baseline value of 74 (20) s to 35 (6) s in the cadaver group and to 56 (16) s in the manikin group. The effect of 'time' was significant (p = 0.002), indicating that both methods of training led to improvements. The training effect of the cadaveric method was greater than with the manikin method (p = 0.0016). Thirty-four failed intubations occurred at baseline vs. eight at the end of study (RR 0.24, 95%CI 0.11-0.51, p = 0.0002, NNT 9.6); six in the cadaver group and two in the manikin group (p = 0.22). We conclude that human cadavers preserved using Thiel's method are potentially better for teaching flexible fibreoptic tracheal intubation compared with manikins.

Suitability of a preserved human cadaver model for the simulation of facemask ventilation, direct laryngoscopy and tracheal intubation: a laboratory investigation.

BACKGROUND: Using fresh or formalin-embalmed cadavers has not been generally accepted for the purposes of teaching airway management. We investigated whether cadavers 'preserved according Thiel's embalming method' (PATEM) are suitable for the simulation of facemask ventilation and tracheal intubation by direct laryngoscopy. METHODS: This observational cluster sampling, controlled simulation study, included eight PATEM cadavers and eight manikins in two clusters. Twenty experienced anaesthetists were randomly assigned to execute 80 facemask ventilations and 80 tracheal intubations in both groups. The ease of facemask ventilation was the primary endpoint. The secondary endpoint was the composite outcomes of laryngoscopy and tracheal intubation. RESULTS: The success rate at the first attempt at mask ventilation was 74% (59/80 attempts) on cadavers and 41% (33/80 attempts) on manikins (P<0.0001). Twenty one subjects received an oral airway in both groups and succeeded in facemask ventilation 20 times on cadavers and four times on manikins (P=0.004). Two-handed technique mask ventilation was required 24 times on manikins and once on cadavers (P=0.0016). In one attempt on a manikin the mask ventilation was impossible. Poor laryngeal view (Cormack-Lehane grade 3) occurred 14 times among cadavers (17.5%) and once in manikins (1.25%) (P=0.007), whereas difficulties in tracheal intubation were encountered 16 times in cadavers (20%) vs 17 times in manikins (21.25%) (P=0.84). In a subjective evaluation the participants preferred the cadaver model over the manikins (P<0.0001). CONCLUSIONS: PATEM cadavers were better suited for facemask ventilation and provided a more realistic environment for laryngoscopy and tracheal intubation than the studied manikins.

Effect of PACAP on LH release studied by cell immunoblot assay depends on the gender, on the time of day and in female rats on the day of the estrous cycle.

We have previously demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) can be released from cultured rat anterior pituitary cells and when added to the medium in physiological concentration it releases LH from individual gonadotropes. In the present work, we studied whether the release of PACAP and the responsiveness of LH cells to PACAP depend on the gender, on the time of day when the animals were sacrificed, and in females on the stage of the estrous cycle. Anterior pituitary cells were cultured on nitrocellulose membrane. We found that the number of PACAP releasing cells was higher in proestrous than in diestrous female or in male rats and their number was always higher in the evening than at the other times. The effect of PACAP on LH cells was stimulatory in the morning of proestrus and diestrus. In proestrous rats, PACAP did not influence LH release in the afternoon or the evening, but in diestrous rats it decreased it in the afternoon and the evening. In males, there was a decrease of LH due to PACAP treatment at 10 and 20 h; however, PACAP did not influence LH at 16 h. It was concluded that in vivo PACAP might be involved in the circadian and episodic release of LH at pituitary level.

Cell immunoblot assay study demonstrating the release of PACAP from individual anterior pituitary cells of rats and the effect of PACAP on LH release.

The presence of pituitary adenylate cyclase activating polypeptide (PACAP) was previously demonstrated in the anterior pituitary by radioimmunoassay, immunohistochemistry, and reverse transcript-polymerase chain reaction (RT-PCR). With the use of cell immunoblot assay (CIBA), when the pituitary cells were cultured on nitrocellulose membrane, the release of PACAP by individual anterior pituitary cells was observed. The released peptide, trapped by the nitrocellulose membrane forming a blot around the cells, was demonstrated by immunocytochemistry. Double labeling revealed that a part of PACAP-immunoreactive cells can release LH as well. With the use of sandwich enzyme immunoassay (S-EIA), it was found that the concentration of PACAP in the anterior pituitaries is 10(-10) M. In cell culture in a similar concentration, PACAP stimulated the LH release from female gonadotropes, but did not influence it from male ones. The stimulated release of LH was indicated by the enhancement in the diameter of LH blots compared to the untreated control cultures. We concluded that PACAP may be released from the anterior pituitary cells in a concentration which would be able to influence LH release not only in vitro but under in vivo conditions as well. The effect of PACAP on LH release was different in female and male pituitary cultures.

Prolactin-synthesizing and prolactin-releasing activity of fetal and early postnatal rat pituitaries: in vivo and in vitro studies using RIA, reverse hemolytic plaque assay and immunocytochemistry.

In vivo and in vitro prolactin (PRL)-synthesizing and PRL-releasing activity of fetal (days 12-22) and early postnatal (days 1-10 after birth) rat pituitaries were studied by means of radioimmunoassay (RIA), reverse hemolytic plaque assay and immunocytochemistry. Using RIA, PRL could first be detected, both in the pituitary and in the serum, on day 17 of fetal development. From this day on, pituitary PRL gradually increased, the rise was particularly marked during the postnatal period and became depressed for the first 10 days of postnatal life. On fetal day 18, 12-15% of monodispersed pituitary cells displayed PRL immunopositivity, but only 3-5% of PRL-positive cells were plaque-forming, i.e. released PRL. By the end of gestation 19-25% and on postnatal day 10 42-45% of all pituitary cells were PRL cells and 31-35 and 15-17% of PRL-positive cells, respectively released PRL. Both pre- and postnatal PRL cells in monolayers were insensitive to TRH treatment. Pituitary primordia immunocytochemically and radioimmunologically negative for PRL (13- to 14-day-old fetal) when placed in serum-free organ culture were able to synthesize and release PRL. Fetal pituitary exhibited a highly regular increasing pattern of daily PRL release during a 7-day-culture period. Data obtained both in vivo and in vitro did not exhibit any sex differences. The present findings are consistent with all those observations suggesting an early emergence of fetal rat pituitary lactotrophs. The in vitro results support the concept that Rathke's pouch cells have substantial degree of independence from extrapituitary regulatory actions in the expression and further progression of specific functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of intratesticular administration of anti-corticotropin-releasing factor antiserum (a-CRF) on testicular function in neonatal rats.

The possible physiological role of testicular corticotropin-releasing factor (CRF) in the regulation of testicular functions was studied in neonatal rats. Two microlitres of anti-CRF-antiserum (dilution: 1:10 or 1:100) was injected intratesticularly to 5 d-old rats with two testes and to hemicastrates. Five days after hemicastration and treatment of the remaining testis with the antiserum, serum testosterone concentration and basal testosterone secretion in vitro decreased significantly. Unilateral testicular injection of a-CRF in rats with two testes resulted in a significant drop in serum testosterone level with no change in basal testosterone production. Data indicate that in neonatal rats testicular CRF might be a local stimulator of steroidogenesis.

Vasectomy modulates the local effect of intratesticular opioids on the gonad of immature rats.

Previous studies indicate that 7 days after testicular administration of naloxone, serum testosterone (T) concentration and basal T secretion in vitro decrease significantly. In neonatal rats, short-term (2 h) intratesticular treatment with 0.3 micrograms (D-Met2-Pro5)-enkephalinamide suppresses steroidogenesis. In the present study, testicular treatment with naloxone or enkephalinamide was combined with hemivasectomy (which also includes transection of the inferior spermatic nerve). When local treatment with naloxone was combined with vasectomy in 15-day-old rats, the opioid antagonist-induced increase in testicular weight, the decrease in basal T secretion of the treated gonad and the drop in serum T level could not be observed 7 days postsurgery, despite the fact that in immature rats, hemivasectomy, by itself, also reduces steroid secretion. Similarly, in 5-day-old animals, the short-term (2 h) effect of testicular enkephalinamide on T secretion was prevented by vasectomy. These data suggest that local testicular actions exerted by endogenous opioid peptides might be modulated by the inferior spermatic nerve.

Fetal pituitary graft is capable of initiating hormone synthesis in median eminence removed adult rat.

Rathke's pouches of 12- and 13-day-old rat embryos were implanted beneath the kidney capsule of adult male rats subjected to the removal of median eminence or to sham-operation. Host animals were sacrificed 28 days after grafting and the implanted pituitaries were processed for immunohistological examination. ACTH, LH-beta, FSH-beta, TSH-beta, GH and PRL immunopositive cells could be observed in fetal grafts of all experimental groups. However, the number and staining intensity of different hormone containing cells largely varied and presumably depended on the hormonal state of host animals. The results indicate that undifferentiated fetal pituitary does not require hypothalamic hypophysiotrophic neurohormones for proliferation and cytodifferentiation and that its development might be modulated by circulating trophic hormones of host animals.

Intratesticular injection of [D-Met2-Pro5]enkephalinamide suppresses testosterone secretion of the testis of immature rat.

The possible role of enkephalin in the local control of testicular function was studied in neonatal rats. 5- and 10-day old hemicastrated rats were treated intratesticularly with an enkephalin analog [D-Met2-Pro5]enkephalinamide. In 5-day-old rats local injection of different doses (0.1-0.3 micrograms/testis) of the peptide suppressed basal testosterone secretion in vitro in a dose-dependent manner 2 h posttreatment. Intratesticular administration of naloxone prior to enkephalin treatment prevented the decrease in basal testosterone production induced by the opioid agonist. In 10-day-old animals intratesticular injection of 1.0 and 3.0 micrograms/testis of enkephalinamide reduced serum testosterone concentration and basal testosterone secretion in vitro. Systemic injection of the peptide produced no change in steroidogenesis. These results suggest that enkephalins might be among the intratesticular factors regulating Leydig cell functions.

Effect of simultaneous local injection of 6-hydroxydopamine and naloxone on the testis of neonatal rats.

The effect of simultaneous intratesticular injection of the opiate receptor antagonist naloxone and the neurotoxic drug 6-hydroxydopamine (6-OHDA) on testicular growth, compensatory testicular hypertrophy, serum testosterone level and basal testosterone secretion in vitro was studied in neonatal rats. In animals with two testes unilateral intratesticular administration of naloxone alone enhanced, while 6-OHDA alone decreased the weight of the treated gonad. In animals treated simultaneously with these two agents the decrease in testicular weight induced by 6-OHDA was partially prevented by naloxone. In hemicastrated animals intratesticular treatment with naloxone enhanced the extent of compensatory testicular hypertrophy. Treatment of the remaining testis with 6-OHDA + naloxone did not interfere with the diminished compensatory testicular hypertrophy observed following 6-OHDA treatment. Data indicate that naloxone can counteract the degenerative effect of 6-OHDA in animals with two testes but not in hemicastrates.

Cultured fetal rat pituitaries kept in synthetic medium are able to initiate synthesis of trophic hormones.

An immunohistochemical study was performed to determine the capacity of early fetal pituitaries to differentiate into specific hormone-synthesizing tissue in the absence of any influence from the central nervous system. Rathke's pouches from rats were removed from their juxtadiencephalic position on day 11 and 12 of gestation and maintained for 2-7 days in a chemically defined culture medium (M 199) without antibiotics and serum supplementation. The immunocytochemical observations provided evidence for the differentiation of ACTH-, TSH-beta-, LH-beta-, FSH-beta-, GH- and PRL-synthesizing cells in the isolated organ cultured from 11 to 12-day-old pituitary primordia. The appearance of specific hormone-synthesizing cells in vitro displayed a delay of 1.5-2 days compared to the day of appearance in vivo, however, the sequential order of developmental events occurred as observed in vivo. The present results suggest that endocrine or neuroendocrine signals are not required for the expression of specific secretory functions of fetal pituitaries, at least at an age of 11-12 days.

Ontogenesis of the three parts of the fetal rat adenohypophysis. A detailed immunohistochemical analysis.

The sequential changes in the histological pattern of anterior pituitary cytodifferentiation of the rat are described. The first labeled cells were ACTH positive and were detected in the pars tuberalis on postconceptual day 13. On day 14 ACTH cells also appeared in the ventral periphery of the pars distalis. On fetal day 15 the pars tuberalis anlage was characterized by numerous well-stained ACTH cells and by some weakly labeled FSH-beta, LH-beta, TSH-beta, GH and PRL cells while the pars distalis showed only ACTH positivity. On day 16 of gestation the ACTH cells were equally distributed throughout the whole pars distalis, while LH-beta, FSH-beta, TSH-beta, PRL and GH immunoreactive cells were localized either in the ventral region of the pars distalis only or were evenly distributed throughout the pars distalis. The present immunocytochemical data suggest that in the pars distalis the hypophyseal cell differentiation follows a clear rostrocaudal, ventrodorsal direction and that the time sequence of the functional differentiation of the adenohypophysis is pars tuberalis, pars distalis and pars intermedia.

[Ontogenesis of the hypothalamo-hypophyseal system. Partial autonomy of the development of the fetal adenohypophysis in the rat]. / Ontogenèse du système hypothalamo-hypophysaire. Autonomie partielle du développement de l'adénohypophyse foetale chez le rat.

Specific differentiation of the hypothalamus and that of hypophysis has been followed. From day 12 of fetal life the synthesis of hypothalamic LHRH could be demonstrated by radioimmunoassay. The presence of adenohypophyseal LHRH receptors was also evident from day 16 of gestation. Studies on gonadotroph hormone synthesis have revealed that production of LH starts on fetal day 12, while that of FSH appears later, on fetal day 19. In order to study the capacity of hypophysis to differentiate, Rathke's pouches removed on fetal days 11-12 were maintained in organ culture. It has been demonstrated by immunocytochemistry and RIA that hormone production occurs also in cultures pituitaries kept in chemically defined medium (M 199). In sum, these results suggest that hypothalamus factors do not seem to play a determining role in the cytodifferentiation of hypophyseal cells.

Hypothalamic GnRH and pituitary gonadotroph relationships during rat fetal life.

The ontogeny of hypothalamic GnRH, of pituitary and gonadal receptors and of pituitary LH and FSH was studied in the fetal and neonatal rat. Hypothalamic, hypophyseal and gonadal primordia were dissected from animals ranging in age from postconceptual day 12 to birth. Immunoreactive GnRH was detectable in the hypothalamus from fetal day 12 onwards at a low level until day 17, whereafter hypothalamic GnRH content and concentration increased until birth. GnRH receptors were reliably detectable in the pituitary anlage from fetal day 16 onwards and increased progressively with advancing age whether expressed as content or concentration. Signs of pituitary LH synthesis were evident as early as fetal day 12 but intrapituitary LH levels remained low until fetal day 17 when levels increased progressively until the end of gestation. Pituitary FSH was undetectable until fetal day 19, thereafter rising dramatically until the end of gestation. GnRH binding to testicular and ovarian tissues was undetectable throughout the period of fetal development. The possible relations among the developmental changes in hypothalamic GnRH, pituitary GnRH receptors and gonadotrophins are discussed.

The effect of neonatal vasectomy on testicular function.

The effect of unilateral or bilateral vasectomy on testicular weight and basal testosterone production in vitro was studied in neonatal intact and hemicastrated rats. Five days after right-side vasectomy ipsilateral to vasectomy testicular weight increased, and basal testosterone production decreased. Ten days postvasectomy the changes were opposite, and affected both testes. In hemicastrated animals hemivasectomy did not interfere with compensatory hypertrophy but induced a significant decrease in basal testosterone production. The data of the present study suggest that in neonatal animals intact ductus deferens bundle(s) is also required for the full control of testicular weight and basal testosterone secretion.

Unilateral vagotomy inhibits compensatory kidney growth after unilateral nephrectomy in rats.

The effect of unilateral vagotomy on compensatory renal growth that follows unilateral nephrectomy was studied. Unilateral vagotomy inhibited the usual compensatory growth of the remaining kidney. Unilateral vagotomy did not effect kidney weight in animals with two kidneys. Data indicate that the vagus nerve is involved in the development of compensatory kidney hypertrophy.

Effect of unilateral or bilateral thyroidectomy on TRH content of hypothalamus halves.

The effect of uni- or bilateral thyroidectomy on the thyrotropin-releasing hormone (TRH) content of the mediobasal hypothalamus (MBH) of the two sides was studied in male rats. Both right- and left-side hemithyroidectomy resulted in a significantly increased TRH content in both halves of the MBH. In addition, the total TRH content rise of the MBH was higher after unilateral than after bilateral thyroidectomy. The mechanism of action of uni- or bilateral thyroidectomy on the hypothalamic TRH content is discussed.

Developmental changes of thyroliberin (TRH) in the rat brain.

Levels of thyroliberin (TRH) were measured by radioimmunoassay in the hypothalamus and in the extrahypothalamic brain tissue of rats from fetal day 12 up to day 47 of postnatal life. TRH was detectable as early as on day 12 of intrauterine life in both the hypothalamic primordia and the extrahypothalamic brain tissue. The increase of hypothalamic TRH content was marked between fetal days 12 and 15, then up to the end of gestation it exhibited a slower tendency of increase. From fetal day 15 up to postnatal day 2 the TRH content of the hypothalamus was always higher than that of the extrahypothalamic brain. Both hypothalamic and extrahypothalamic TRH content increased up to day 35 of postnatal life.

Depending on the dose 6-OHDA stimulates or inhibits the testis of immature rats.

The effect of the neurotoxic drug, 6-hydroxy-dopamine (6-OHDA) on the testicular growth, the compensatory testicular hypertrophy, the testicular cytology and the serum testosterone level was studied in immature rats. Unilateral injection of 33 micrograms of 6-OHDA beneath the testicular capsule in animals with two testes did not alter the weight of the gonads but promoted the development of both testes. Unilateral injection of 333 micrograms of 6-OHDA resulted in a decreased weight and severe atrophy of the treated gonad, while a significant weight gain of the contralateral testis occurred. In hemicastrated rats 33 micrograms of 6-OHDA into the remaining testis caused an enhanced compensatory testicular hypertrophy. When 333 micrograms of 6-OHDA was administered beneath the capsule of the remaining testis a diminished compensatory testicular hypertrophy occurred but no signs of degeneration could be observed. Data indicate that the testicular adrenergic elements play a role in the testicular growth, in the development of compensatory testicular hypertrophy and in the cytodifferentiation of the immature testis.