Is propranolol compounding from tablet safe for pediatric use? Results from an experimental test.

AIM: Although propranolol is widely used in the treatment of infantile hemangiomas, the standard 40 mg tablet needs to be fractioned to obtain 10 mg parts, with even lower doses (i.e., 2-3 mg/kg/day divided into 2-3 daily doses) required in infants. This study evaluated the weight and dose uniformity in split quarters of propranolol tablets. METHODS: Twenty pharmacy students split 70 propranolol tablets by using a kitchen knife in order to obtain 200 quarters, which were considered integral and adequate for administration. Intact tablets and quarters were weighed. The content of propranolol in tablet quarters was determined on 200 quarters by using high performance liquid chromatography. RESULTS: Overall, 265 parts (94.6%) were integral and 213 (76.1%) were considered as adequate for administration. The mean (± standard deviation) weight of quarters judged as suitable and non-suitable for administration was 49.56 ± 5.27 mg and 46.24 ± 7.53 mg, respectively. Splitting caused a mean weight loss in each tablet of 2.97 ± 2.91 mg (median 2.06 mg). The percentage of quarters with weight lower than theoretical was 55.88%, and the remaining weighted more than expected. The mean propranolol content in quarters was 9.52 ± 0.96 mg (median 9.42 mg, range 7.36-12.23 mg) and 42% of quarters were out of the ± 10% acceptance range. CONCLUSION: The manual splitting of propranolol 40 mg tablets produced a significant proportion of quarters not suitable for administration in children or with a weight and/or an active concentration outside of the required range. The availability of a pediatric oral solution of propranolol will reduce the risk of incorrect dosing.

Immunohistochemical evidence for the presence of various kisspeptin isoforms in the mammalian brain.

Kisspeptins are small peptides encoded by the Kiss1 gene that have been the focus of intense neuroendocrine research during the last decade. Kisspeptin is now considered to have important roles in the regulation of puberty onset and adult oestrogen-dependent feedback mechanisms on gonadotrophin-releasing hormone secretion. Several kisspeptin antibodies have been generated that have enabled an overall view of kisspeptin peptide distribution in the brain of many mammalian species. However, it remains that the distribution of the different kisspeptin isoforms is unclear in the mammalian brain. In the present study, we report on two new N-terminal-directed kisspeptin antibodies, one against the mouse kisspeptin-52 sequence (AC053) and one against the rat kisspeptin-52 sequence (AC067), and use them to specifically map these long isoforms in the brains of mouse and rat, respectively. Kisspeptin-52 immunoreactivity was detected in the two main kisspeptin neuronal populations of the rostral periventricular area and arcuate nucleus but not in the dorsomedial hypothahamus. A large number of fibres throughout the ventral forebrain were also labelled with these two antibodies. Finally, a comparison with the most commonly used C-terminal-directed kisspeptin antibodies further suggests the presence of shorter kisspeptin fragments in the brain with specific inter- and intracellular expression patterns.

Embryonic development of kisspeptin neurones in rat.

Kisspeptins, encoded by the Kiss1 gene, play a key role in the regulation of reproductive function, although very little is known about the ontogenesis of this system. The present study aimed to determine the period of arcuate nucleus (ARC) kisspeptin cell birth and the embryonic stage and neuroanatomical sites of onset of kisspeptin immunoreactivity. Bromodeoxyuridine (BrdU) was administered to female rats at various gestational stages and double immunohistochemistry against kisspeptin and BrdU was performed on brain sections from their offspring. The period of neurogenesis of ARC kisspeptin neurones begun between embryonic day (E) 12.5 and E13.5, reached its peak at E15.5 and was not completely over at E17.5. Kiss1 mRNA was detected in mediobasal hypothalamic punches of embryos aged E14.5, E16.5, E18.5 and E22.5 by real-time reverse transcriptase-polymerase chain reaction. Accordingly, kisspeptin-immunoreactive (-IR) cells were consistently detected in the embryonic ARC from E14.5 and their number increased until E18.5 to reach approximately half the level observed in adults. Between E18.5 and E22.5, the number of kisspeptin-IR cells and hypothalamic Kiss1 expression significantly decreased, regardless of sex, and this decrease persisted until birth. Taken together, these results demonstrate that rat ARC kisspeptin neurones are born locally during an extended embryonic period and are able to synthesise kisspeptins rapidly after their birth, consistent with the hypothesis of a role during embryonic activation of the hypothalamic-hypophyseal-gonadal axis. A sex-independent decrease of kisspeptin-IR cell numbers was observed during the perinatal period, suggestive of important regulations of kisspeptin neurones around birth.

Kisspeptin-immunoreactivity changes in a sex- and hypothalamic-region-specific manner across rat postnatal development.

Kisspeptins are potent secretagogues of gonadotrophin-releasing hormone, playing a key role in puberty onset. These peptides are produced by distinct neuronal populations of the hypothalamus located in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (ARC). The present immunohistochemical study aimed to determine the spatiotemporal onset of kisspeptin-immunoreactivity (-IR) in the neonatal hypothalamus of male and female rats and to evaluate changes in kisspeptin-IR around puberty. Kisspeptin-IR cells and fibres could be detected from the day of birth in the ARC of both males and females. At this stage, only females displayed some kisspeptin-IR fibres in the RP3V. From postnatal day 7 to adulthood, males displayed lower levels of kisspeptin-IR than females in both regions. During infancy, kisspeptin-IR fibre density in the female decreased in the ARC, whereas it increased in the RP3V. A sex-independent decline in RP3V kisspeptin-IR fibre density was observed in the juvenile, followed by a peripubertal increase in RP3V and ARC kisspeptin-IR. These peripubertal increases in kisspeptin-IR occurred at different timings dependent on sex and region. In females specifically, the increase in kisspeptin-IR fibre density occurred first in the ARC and later in the RP3V under constant levels of circulating oestradiol. In conclusion, the present study highlights the expression of hypothalamic kisspeptins soon after birth, as well as the neonatal establishment of a strong and persisting sex difference in ARC kisspeptin-IR in rats. Moreover, a female-specific desynchronisation of the ARC and RP3V was observed with respect to the increase in kisspeptin-IR fibre density around puberty, which was not related to peripubertal variations in circulating oestradiol.

Evidence for cell proliferation in the sheep brain and its down-regulation by parturition and interactions with the young.

Production of new neurons continues throughout life in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus and is influenced by both endocrine and social factors. In sheep parturition is associated with the establishment of a selective bond with the young based on an olfactory learning. The possibility exists that endocrine changes at parturition together with interactions with the young modulate cell proliferation in the neurogenic zones. In the present study, we first investigated the existence of cell proliferation in sheep. Newly born cells labeled by the cell proliferation marker 5-bromo-2'-deoxyuridine (BrdU) were found in the SVZ, the main olfactory bulb (MOB) and the DG and completely co-localized with Ki-67, another mitotic marker. Forty to 50% of the BrdU-labeled cells contained GFAP suggestive of the presence of neural stem cells. Secondly, parturition with or without interactions with the lamb for 2 days, down-regulated the number of BrdU-labeled cells in the 3 proliferation sites in comparison to no pregnancy. An additional control provided evidence that this effect is specific to early postpartum period: estrus with interactions with males did not affect cell proliferation. Our results provide the first characterization of neural cell proliferation in the SVZ, the DG and unexpectedly in the MOB of adult sheep. We hypothesize that the down-regulation of cell proliferation observed in the early postpartum period could facilitate the olfactory perceptual and memory demands associated with maternal behavior by favouring the survival and integration of neurons born earlier.

Mapping of kisspeptin fibres in the brain of the pro-oestrous rat.

Kisspeptins are a family of small peptides that play a key role in the neuroendocrine regulation of the reproductive function through neural pathways that have not yet been completely identified. The present study aimed to investigate the distribution of kisspeptin neurone fibres in the female rat brain by comparing precisely the immunoreactive pattern obtained with two antibodies: one specifically directed against kisspeptin-52 (Kp-52), the longest isoform, and the other directed against kisspeptin-10 (Kp-10), whose sequence is common to all putative mature isoforms. With both antibodies, immunoreactive cell bodies were exclusively observed in the arcuate nucleus, and immunoreactive fibres were confined to the septo-preoptico-hypothalamic continuum of the brain. Fibres were observed in the preoptic area, the diagonal band of Broca, the septohypothalamic area, the anteroventral periventricular, suprachiasmatic, supraoptic, paraventricular and periventricular nuclei, the dorsal border of the ventromedian nucleus, the dorsomedial and arcuate nuclei, and the median eminence. In the latter structure, varicose fibres were mainly distributed in the internal layer and were detected to a lesser extent throughout the external layer, including around the deeper part of the infundibular recess. Most regions of immunoreactive cells and fibres matched perfectly for the two antibodies. However, fibres in the dorsolateral septum, anterior fornix, accumbens nucleus and the lateral bed nucleus of the stria terminalis were only recognised by antibody anti-Kp-10, suggesting that anti-Kp-10 may recognise a wider range of kisspeptin isoforms than anti-Kp-52 or cross-react with molecules other than kisspeptin in rat tissue. Overall, these results illustrate the variety of projection sites of kisspeptin neurones in the rat and suggest that these peptides play a role in different functions.

Kisspeptin and the preovulatory gonadotrophin-releasing hormone/luteinising hormone surge in the ewe: basic aspects and potential applications in the control of ovulation.

The identification of the neural mechanisms controlling ovulation in mammals has long been a 'holy grail' over recent decades, although the recent discovery of the kisspeptin systems has totally changed our views on this subject. Kisspeptin cells are the major link between gonadal steroids and gonadotrophin-releasing hormone (GnRH) neurones. In the female rodent, kisspeptin cells of the preoptic area are involved in the positive-feedback action of oestrogen on GnRH secretion, although the picture appears more complicated in the ewe. As in rodents, activation of preoptic kisspeptin neurones accompanies the GnRH surge in the ewe but an active role for arcuate kisspeptin neurones has also been proposed. Experimentally, kisspeptin is able to restore reproductive function when the hypothalamic-hypophyseal ovarian axis is quiescent. For example, i.v. infusion of a low dose of peptide in anoestrous ewes induces an immediate and sustained release of gonadotrophin, which subsides and then provokes a luteinising hormone (LH) surge a few hours later. This pharmacological intervention induces the same hormonal changes normally observed during the follicular phase of the oestrous cycle, including the secretion of oestrogen and its negative- and positive-feedback actions on the secretion of LH and follicle-stimulating hormone. Accordingly, a high percentage of kisspeptin-infused animals ovulated. Although the multiple facets of how the kisspeptin systems modulate GnRH secretion are not totally understood, the demonstration that exogenous kisspeptin administration can induce ovulation in anovulatory animals paves the way for future therapeutic applications aiming to control reproduction.

Kisspeptin immunoreactive neurons in the equine hypothalamus Interactions with GnRH neuronal system.

To determine if kisspeptin could be implicated in the control of reproduction in equine species, we studied the distribution of kisspeptin neurons and their anatomical interactions with GnRH neurons in the hypothalamus of pony mares. Brains were collected in three pony mares between 2 and 4h after ovulation. One major population of kisspeptin immunoreactive cell bodies was found in the arcuate nucleus (ARC), where they extended from the middle of the nucleus to the premammillary recess. Kisspeptin immunoreactive varicose fibers extended from the preoptic area to the mammillary nuclei, with important densities especially in the anterior periventricular area and the median eminence (ME). Rare close appositions of kisspeptin fibres on GnRH cell bodies were observed in the ARC. Close appositions between kisspeptin and GnRH fibres were also confirmed at a low incidence in the anterior basal periventricular area and at a high incidence in the ME. This work provides neuroanatomical bases for further investigations into the role of kisspeptin in equine reproduction.

Basic aspects of the control of GnRH and LH secretions by kisspeptin: potential applications for better control of fertility in females.

The neuronal control of fertility and sterility has been a subject of research for years. However, nowadays, in spite of considerable literature about GnRH during the last few decades, the precise cellular and molecular mechanisms whereby gonadal steroids and other peripheral signals converge in the brain to achieve the fine regulation of GnRH secretion remains partially unknown. In this scenario, a major breakthrough in our understanding of the neuronal signals governing reproduction took place in 2003 with the discovery of metastin/kisspeptin as a major player in the control of GnRH secretion. This molecule, first described as having a crucial role in triggering the onset of puberty, is involved in all phases of reproductive life and hence has attracted the interest of many reproductive neuroendocrinologists. Administered either centrally or peripherally, kisspeptin strongly induces the secretion of gonadotropin in many species, mainly through stimulation of GnRH secretion. Kisspeptin cells involved in the control of GnRH secretion are located in two regions of the brain: the preoptic area and the arcuate nucleus. Carrying oestradiol receptor alpha, kisspeptin cells of these regions appear to be the main integration centres for the expression of both the positive and negative feedback of steroid on GnRH secretion. More recently, this molecule has been shown to be able to synchronize preovulatory surges in cyclic ewes and cause ovulation in seasonally acyclic ewes. This review summarizes the most relevant aspects of the role of kisspeptin in GnRH/LH release and the potential application of this molecule in new strategies for controlling female fertility.

Kisspeptin immunoreactive cells of the ovine preoptic area and arcuate nucleus co-express estrogen receptor alpha.

Kisspeptins are peptide ligands of the G protein-coupled receptor GPR54, recently shown to be essential to reproductive function. We have raised specific rabbit antisera against a highly conserved 10 amino acid-amidated peptide (kp10) common to all kisspeptin isoforms isolated so far and mapped the distribution of kp10-immunoreactive (ir) cells in the ovine hypothalamus. Kp10-ir cells were predominant in the caudal arcuate nucleus, the dorsomedial nucleus and the medial preoptic area. Numerous varicose kp10-ir fibers were found in the preoptic area where GnRH neurons reside and in the median eminence, seemingly projecting around small capillaries in its external zone. Within the caudal arcuate nucleus, nearly all kp10-ir cells showed an intense estradiol receptor alpha immunofluorescent signal compared with approximately half of kp10-ir cells in the preoptic area. The pattern of distribution of kp10 immunoreactivity in the hypothalamus suggests a role for kisspeptin in the estrogen-dependent regulation of GnRH and LH secretion in the ewe.

Efficient gene transfer in mouse neural precursors with a bicistronic retroviral vector.

Gene transfer into neural precursors is a powerful approach to study the function of specific gene products during nervous system development. Here we describe a retrovirus-based methodology to transduce foreign genes into mouse neural precursors. We used a high-titer bicistronic retroviral vector that encodes a marker gene, placental alkaline phosphatase (plap), and a selection gene, neomycin phosphotransferase II (neoR), under the translational control of two retroviral internal ribosome entry segments. Transduction efficiency even without selection was up to 95% for multipotential neurospheres derived from embryonic striata and grown with basic fibroblast growth factor 2. Expression of plap and neoR was sustained with time in culture and upon differentiation into neurons, astrocytes, and oligodendrocytes, as shown by double immunofluorescence labeling with cell type-specific markers, Western blotting, and neomycin resistance. However, levels of plap were decreased in differentiated oligodendrocytes. Transduction with the same vector of neonatal oligodendrocyte precursors grown in oligospheres consistently resulted in a lower proportion of plap-immunoreactive cells and enhanced cell death in the absence of neomycin. However, plap expression was maintained in some differentiated oligodendrocytes expressing galactocerebroside or myelin basic protein. In that neurospheres can be easily expanded in vitro and factors enabling their differentiation into the three main central nervous system cell types are being elucidated, this methodology could be used in the future to produce large number of transduced, differentiated neural cells.

Polysialyltransferase ST8Sia II (STX) polysialylates all of the major isoforms of NCAM and facilitates neurite outgrowth.

The neural cell adhesion molecule (NCAM) has different isoforms due to different sizes in its polypeptide and plays a significant role in neural development. In neural development, the function of NCAM is modified by polysialylation catalyzed by two polysialyltransferases, ST8Sia II and ST8Sia IV. Previously, it was reported by others that ST8Sia II polysialylates only transmembrane isoforms of the NCAM, such as NCAM-140 and NCAM-180, but not NCAM-120 and NCAM-125 anchored by a glycosylphosphotidylinositol. In the present study, we first discovered that ST8Sia II polysialylates all isoforms of the NCAM examined, and we demonstrated that polysialylation of NCAM expressed on 3T3 cells facilitates neurite outgrowth regardless of isoforms of NCAM, where polysialic acid is attached. We then show that neurite outgrowth is significantly facilitated only when polysialylated NCAM is present in cell membranes. Moreover, the soluble NCAM coated on plates did not have an effect on neurite outgrowth exerted by soluble L1 adhesion molecule coated on plates. These results, taken together, indicate that ST8Sia II plays critical roles in modulating the function of all major isoforms of NCAM. The results also support previous studies showing that a signal cascade initiated by NCAM differs from that initiated by L1 molecule.

Developmental pattern of expression of the alpha chemokine stromal cell-derived factor 1 in the rat central nervous system.

Stromal cell-derived factor 1 (SDF-1) is an alpha-chemokine that stimulates migration of haematopoietic progenitor cells and development of the immune system. SDF-1 is also abundantly and selectively expressed in the developing and mature CNS, as we show here. At embryonic day 15, SDF-1 transcripts were detected in the germinal periventricular zone and in the deep layer of the forming cerebral cortex. At birth, granule cells in the cerebellum and glial cells of the olfactory bulb outer layer showed an SDF-1 in situ hybridization signal that decreased progressively within the next 2 weeks. In other regions such as cortex, thalamus and hippocampus, SDF-1 transcripts detected at birth progressively increased in abundance during the postnatal period. SDF-1 protein was identified by immunoblot and/or immunocytochemistry in most brain regions where these transcripts were detected. SDF-1 was selectively localized in some thalamic nuclei and neurons of the fifth cortical layer as well as in pontine and brainstem nuclei which relay the nociceptive response. The presence of SDF-1 transcripts in cerebellar granule cells was correlated with their migration from the external to the inner granular layers with disappearance of the signal when migration was completed. In contrast, SDF1 mRNA signal increased during formation of the hippocampal dentate gyrus and stayed high in this region throughout life. The selective and regulated expression of SDF-1 in these regions suggests a role in precursor migration, neurogenesis and, possibly, synaptogenesis. Thus this alpha chemokine may be as essential to nervous system function as it is to the immune system.

Neuregulin is a mitogen and survival factor for olfactory bulb ensheathing cells and an isoform is produced by astrocytes.

The rat olfactory bulb is an exceptional CNS tissue. Unlike other areas of the brain, growing axons are able to enter the olfactory bulb and extend within this CNS environment throughout adult life. It appears that the glial cells of the olfactory system, known as olfactory bulb ensheathing cells (OBECs), may have an important role in this remarkable process of CNS neural regeneration. OBECs are unusual glial cells, possessing properties of both astrocytes and Schwann cells. In this study we show that astrocytes (in the form of astrocyte-conditioned medium; ACM) produce two critical regulatory functions for OBECs: mitogenic activity and a survival factor. Interestingly, the ACM-derived activity for OBECs appears to reside in a signalling protein(s) belonging to the neuregulin (NRG) family of growth factors, and specifically appears to coincide with one or more products of the nrg-1 gene. Our observations provide evidence for the following: recombinant human neu differentiation factors (NDFbeta1, -2 and -3) are mitogenic to OBECs; the activity in ACM can be neutralized by NDF antibodies; these same antibodies detect a 50-kDa, non-heparin binding protein in concentrated ACM; astrocytes express detectable nrg-1 transcripts; and OBECs express functional NRG receptors erbB2 and erbB4.

Adhesion molecule expression and phenotype of glial cells in the olfactory tract.

These data illustrate that OBECs have a highly plastic nature in keeping with their need to respond rapidly to changing environmental cues. This relates to their required function in supporting axonal extension throughout life. Future studies using antibodies to PSA-NCAM and L-NGFr together with FACS sorting to purify the two types of OBEC should give us a clearer understanding of the lineage relationship of the two phenotypes. With purified populations of the astrocyte-like and Schwann cell-like OBEC we should be able to determine if these cells have different functions in vivo, using several approaches namely: i) identifying the growth factors that regulate their growth and differentiation, ii) measuring the ability of the purified cells to remyelinate the experimentally-created CNS lesions and iii) carry out more detailed cellular and molecular comparisons of the two phenotypes.

Schwann cell-like myelination following transplantation of an olfactory bulb-ensheathing cell line into areas of demyelination in the adult CNS.

In this study we have transplanted a clonal olfactory bulb-ensheathing cell line into focal areas of the rat spinal cord which contain demyelinated axons but neither oligodendrocytes nor astrocytes. The cell line was created by retroviral incorporation of the temperature-sensitive Tag gene into FACS-sorted 04+ cells from 7-day-old rat pup olfactory bulb. The spinal cord lesions were obtained by injecting small volumes of ethidium bromide into the dorsal white matter of spinal cord previously exposed to 40 Grays of X-irradiation. Many of the axons were remyelinated by PO+ myelin sheaths 21 days after transplantation. Light and electron microscopy revealed cells engaging and myelinating axons in a manner highly reminiscent of Schwann cells within similar lesions. GFAP+ cells were also present within the lesion. This study provides the first in vivo evidence that olfactory bulb-ensheathing cells are able to produce peripheral-type myelin sheaths around axons of the appropriate diameter.

Low-affinity NGF-receptor and E-N-CAM expression define two types of olfactory nerve ensheathing cells that share a common lineage.

Previously, we have shown that the O4 antibody can be used to define and purify olfactory nerve ensheathing cells (ONECs) from the rat olfactory bulb by fluorescence-activated cell sorting. In this study, using a larger panel of neural markers, we demonstrate that this apparently homogeneous population of ONECs possess a heterogeneous antigenic profile both in vivo and in vitro. The antigenic profile of the sorted cells initially correlated with their antigenic profile in vivo, although expression of some of the markers was either lost or gained during time in culture. These changes were influenced by the culture conditions, with a greater loss of "typical" ONEC markers in serum-containing medium. In serum-free medium, which maintains the cells in a phenotype that closely resembles their in vivo counterparts, we were able to reclassify the ONECs into two cell types based on morphology and antigenic phenotype by using antibodies to polysialic acid (correlating with the embryonic form of N-CAM expression) and the low-affinity nerve growth factor receptor. A detailed immunocytochemical study of the developing olfactory system showed that these two cell types could also be detected along the entire length of the olfactory nerve and the outer layer of the olfactory bulb from Embryonic Day 14 to adulthood, suggesting they were not an in vitro artefact. To address the relationship between the two cell types we constructed a clonal ONEC cell line by retroviral infection with the temperature-sensitive mutant gene of the large T antigen. This clonal cell line contained cells that expressed antigenic phenotypes of both classes of ONECs, suggesting that both cell types are related and share a common lineage.

Tratamento cirúrgico da comunicaçäo inter-auricular (CIA) / Surgical treatment of the atrial septal defect (ASD)

A CIA é uma cardiopatia congênita frequente que pode ocasionar manifestaçöes clínicas graves quando näo tratada. A correçäo cirúrgica é feita com baixa mortalidade hospitalar e significa, na maioria dos casos, cura total da lesäo, por isto os pediatras e cardiologistas necessitam ficar atentos para näo deixar a lesÝo passar desapercebida, como frquentemente ocorre. Os autores analisam um grupo de 104 pacientes consecutivos portadores da cardiopatia e que foram operados com baixa mortalidade hospitalar (1,9 por cento) e chamam a atençäo pela frequente presença de lesöes associadas (30 por cento) que também devem ser corrigidas em procedimento cirúrgico único

Successful treatment of a steroid-resistant form of idiopathic thrombocytopenic purpura in pregnancy with high doses of intravenous immunoglobulins.

Idiopathic thrombocytopenic purpura (ITP) during pregnancy may cause serious bleeding in the mother and fetus. Therapy with high-dose intravenous immunoglobulins has caused an immediate and predictable rise in platelet count in both adults and children with chronic or acute ITP. We report our experience in managing a woman near term in pregnancy. The patient demonstrated a rapid increase in platelet count, delivered without excessive bleeding and had a normal child with normal platelet count. Intravenous immunoglobulins may offer a new and safe way to control maternal and fetal platelet counts during pregnancy, delivery and neonatal period.