5th Postnatal Day Research Articles

Mortality after carotid body denervation in rats.

Carotid body denervation (CBD) in neonatal goats and piglets results in minimal irregular breathing and no fatalities. Redundancy and/or plasticity of peripheral chemosensitivity and a relatively mature ventilatory control system at birth may contribute to the paucity of CBD effects in these species. In the present study, we tested the hypothesis that CBD mortality would be greater in neonates of a less mature species such as the rat. We found that the mortality in rats denervated at 2-3 and 7-8 days of age was significantly higher (P < 0.05) than in sham-CBD rats. In all surviving rats, pulmonary ventilation during hypoxia was lower in CBD than in sham operated rats 2 days after denervation. In surviving rats denervated during the 7th and 8th postnatal days, there was also reduced weight gain and pulmonary ventilation during eupnea, including apneas up to 20 s in duration. However, the effects of CBD were compensated within 3 wk after denervation. Local injections of NaCN indicated that aortic chemoreceptors might have been one of the sites of recovery of peripheral chemosensitivity. We concluded that CBD has higher mortality in newborn rats than in other mammals, possibly because of the relative immaturity of these animals at birth. Nonetheless, in survivors there was enough redundancy and plasticity in the control of breathing to eventually compensate for the consequences of CBD.

Analysis of Scar Tissue Distribution on Rat Palates: A Laser Doppler Flowmetric Study

Abstract Objective: The aim of this study was to evaluate the effectiveness of laser Doppler flowmetry (LDF) to discriminate postoperative scar tissue distribution on the palate. Methods: Nineteen male Wistar rats at the 20th postnatal day were divided into experimental and control groups. In the experimental group, lateral palatal mucoperiosteum was excised to form scar tissue on the palate. At the 11th postnatal week, changes in the palatal blood flow were recorded with LDF in both groups by occluding exposed common carotid arteries. Perfusion values of nonoccluded (stable) and occluded states and the ratio of occluded to stable states were compared for scar tissue and normal tissue areas, and also for the normal tissue areas. After the LDF measurements, India ink–injected specimens and tissue sections were prepared for histological observations. Results and Conclusions: Scar tissue areas showed lower perfusion values both in the stable and occluded states, reflecting a lower vascular density in the sca...

Analysis of scar tissue distribution on rat palates: a laser Doppler flowmetric study.

The aim of this study was to evaluate the effectiveness of laser Doppler flowmetry (LDF) to discriminate postoperative scar tissue distribution on the palate. Nineteen male Wistar rats at the 20th postnatal day were divided into experimental and control groups. In the experimental group, lateral palatal mucoperiosteum was excised to form scar tissue on the palate. At the 11th postnatal week, changes in the palatal blood flow were recorded with LDF in both groups by occluding exposed common carotid arteries. Perfusion values of nonoccluded (stable) and occluded states and the ratio of occluded to stable states were compared for scar tissue and normal tissue areas, and also for the normal tissue areas. After the LDF measurements, India ink-injected specimens and tissue sections were prepared for histological observations. Scar tissue areas showed lower perfusion values both in the stable and occluded states, reflecting a lower vascular density in the scar tissue. The ratio of the occluded to stable states was higher in the scar tissue than in the normal tissue areas. In normal tissue areas, perfusion values of both the stable and occluded states appeared to vary, but the ratio did not vary among the areas. In the LDF study, the ratio of the occluded to stable states was considered to be the better parameter for discriminating scar tissue from normal tissue.

Expression of connexin 32 and 43 in developing rat submandibular salivary glands

The expression of the gap-junction proteins connexin 32 and 43 in the developing rat submandibular gland was determined using reverse transcription-polymerase chain reaction and immunohistochemistry. Sprague–Dawley rats from the 17th gestational day through to the 28th postnatal day were used. Connexin 43 gene expression was detected on the 17th gestational day. Connexin 32 gene expression was also detected on the 17th gestational day but less intensely. Immunolabelling for connexin 43 was also found in the developing submandibular gland from the 17th gestational day. Prenatally the most immunoreactive areas for connexin 43 were located in the periphery of terminal tubules, but some staining was discernible between the cells in terminal buds. In the postnatal period, reactive areas were located at both the periphery of acinus-like structures and around the intercalated duct. This is consistent with the known association of connexin 43 with myoepithelial cells. Connexin 32 immunostaining was first detected in the developing submandibular glands on the 18th day of prenatal development. Positive staining was present on the lateral side of the proacinar and mature acinar cells. The number of immunoreactive areas per cell increased during early development followed by a significant decrease perinatally. During postnatal development the density of areas again showed a pattern of increase. These results suggest that connexin 43 is associated with growth and differentiation in the pre- and perinatal periods and also with the contractile function of myoepithelial cells in the postnatal period of the developing submandibular gland. It is also implied that the number of connexin 32-positive areas may correspond to an increase or decrease in the number of proacinar and mature acinar cells.

Postnatal morphodifferentiation of the subneural apparatuses of the posterior cricoarytenoid muscle in rats: a scanning electron microscopy study.

The subneural apparatus, i. e., the post-synaptic component of the neuromuscular junction, in the posterior cricoarytenoid muscle of the rat was studied by scanning electron microscopy, with special attention given to its postnatal differentiation along with the functional development of the muscle. Primitive synaptic troughs observed in the first postnatal week consisted of single cup-like depressions 5-6 microm in diameter. On the 7th day, low sarcoplasmic ridges appeared in the trough. In the second postnatal week, muscle fibers could be classified into two groups: large (10-15 microm in diameter) and small (less than 10 microm in diameter). In the large muscle fibers, many low ridges became circular and protruded to transform the single trough into numerous cup-like depressions (2-5 microm in diameter). In contrast, the subneural apparatus in the small muscle fibers consisted of a small number of cup-like depressions. The two types of subneural apparatus differentiated into adult forms by the 28th postnatal day, although they remained smaller in size than those of adults. In the large muscle fibers, the number of pit-like or elongated invaginations increased and gradually transformed into slit-like junctional folds by the 28th postnatal day, while the small muscle fibers still possessed a few pit-like or elongated junctional folds at this point in time. The two types of morphodifferentiation of the subneural apparatus are thought to reflect the two types of muscle fibers in the rat posterior cricoarytenoid muscle.

Mouse urothelial cells in early postnatal development - proliferation and apical plasma membrane specialization.

The purpose of this work was to investigate proliferation and differentiation of the mouse urothelial cells from the day of birth until the 5th postnatal day. The expression of proliferating cell nuclear antigen (PCNA) was studied immunocytochemically and the differentiation of apical plasma membrane of superficial cells was analysed by scanning microscopy. It was established that proliferation activity is very high during all five days since PCNA positive cells are seen in the superficial and in basal cell layer of the urothelium. Results of scanning microscopy show that the differentiation of superficial urothelial cells is a nonsynchronous process, which gradually progresses from the day of birth and leads into homogeneous population of terminally differentiated superficial cells on the 5th postnatal day.

Vestibular deprivation and the development of dendrite bundles in the rat.

Motoneuronal pools of muscles that subserve postural tasks contain dendrite bundles. We investigated in the rat the development of these bundles in the pools of the long back muscles and related this to postural development. Motoneurons and their dendrites were retrogradely labeled by injecting unconjugated cholera toxin subunit B (CTB) into the muscles of 54 normal rats from birth until adulthood and into 18 rats that were vestibularly deprived from the 5th postnatal day (P5). Dendrite bundles coursing in a transverse direction already occurred at P1. From P4, the first longitudinal bundles could be observed, but the major spurt in development occurred between P6 and P9, when conspicuous bundles developed coursing in rostro-caudal and tranverse directions. This is the age when rats become able to stand freely and walk a few steps. Around P20, the dendrite bundles attained their adult characteristics. Vestibular deprivation by plugging both semicircular horizontal canals did not lead to a retarded development of dendrite bundles nor to a changed morphology. This finding is remarkable, as behavioral analysis showed a delay in postural development by about 3 days. We hypothesize that dendrite bundles in the pools of the long back muscles function to synchronize the motoneurons in different spinal cord segments.

Cell proliferation and death of Hertwig's epithelial root sheath in the rat.

Hertwig's epithelial root sheath (HERS) degenerates immediately after root dentin is formed. However, odontogenic tumors or cysts may originate from residual cells, although little is known about how HERS proliferates and disappears. This study investigated whether cell death is provoked in the tissues surrounding the root during eruption of the rat upper molar. We employed the TdT-mediated-dUTP nick end labeling (TUNEL) method and transmission electron microscopy (TEM) to observe the morphological features of cell death. We examined the activity of cell proliferation immunohistochemically using proliferative cell nuclear antigen (PCNA) and the continuity of HERS using polyclonal keratin antibody (PK). Cell death resembling apoptosis and apoptotic bodies phagocytosed by neighboring mesenchymal cells were detected in only a few cells by both TUNEL and TEM. We also found cells with electron-lucent cytoplasm which contained dilated or ruptured mitochondria and remarkably dilated rough endoplasmic reticulum (rER) which lay sparsely along the root. These cells seemed to be dead HERS cells based on their ultrastructural features, location, and stage. PCNA-positive cells were found in the apical end of the HERS cells, fibroblasts of the periodontal ligament, and odontoblasts. PK reacted with HERS; however, PK-positive cells partially disappeared after the 15th postnatal day when the root dentin had formed slightly. These results may indicate that HERS cells migrate into the periodontal ligament or die immediately after root dentin is formed and that various types of cell death such as apoptosis and cytoplasmic type occur in the tissues surrounding the root during tooth development.

Glial fibrillary acidic protein in the brain of prenatally stressed rats

The content of glial fibrillary acidic protein (GFAP) was studied in the brain structures of rats borne by intact females and females that underwent stress. In the offspring of stressed rats, the GFAP content in the brain gray and white matter on the 15th postnatal day noticeably dropped. On the 30th postnatal day, the GFAP content in the cortex and pons increased, while it somewhat decreased in the striatum and cerebellum. The results suggest that formation of the intermediate astrocyte filaments in the animals subjected to prenatal stress is markedly disturbed.

Postnatal Changes of Leptin Levels in Full-Term and Preterm Neonates: Their Relation to Intrauterine Growth, Gender and Testosterone

The present study was carried out to investigate leptin levels in arterial and venous cord serum and in amniotic fluid in full-term infants at birth and on the 5th postnatal day to define the relationship of leptin to intrauterine growth rate, gender and early postnatal life. The relation of weight gain to serum leptin levels in male preterm infants was determined measuring leptin concentration weekly in the first 5 postnatal weeks. Testosterone levels were determined simultaneously to explore a possible relationship between leptin and testosterone concentrations.Fifty-three term newborn infants with mean birth weight and gestational age of 3,419 g (range 2,150–4,480) and 38.9 weeks (range 36–41) and 19 preterm male infants (mean birth weight and gestational age were 1,416 g (770–1,800) and 30.2 weeks (26–35) were enrolled into the study. Leptin and testosterone levels were determined by radioimmunoassay. It was demonstrated that serum leptin levels were markedly elevated in the cord blood without discernible arteriovenous differences. Cord blood leptin was found to correlate with birth weight (r = 0.40, p < 0.002), weight to length ratio (r = 0.40, p < 0.002) and body mass index (r = 0.35, p < 0.005). It was significantly lower in boys as opposed to girls (p < 0.01) and there was an apparent fall by the 5th postnatal day (p < 0.001). Amniotic fluid contained leptin in much less concentration than cord blood and it proved to be independent of intrauterine growth or gender. Serum leptin concentration in preterm infants at 1 week of age was significantly lower compared with term infants (p < 0.002) and it increased progressively with age (p < 0.01). An inverse relationship was found between leptin and testosterone level (r = –0.358, p < 0.01) and a positive correlation between leptin level and weight/height ratio (r = 0.674, p < 0.01).It is concluded that leptin derived either from placenta or fetal adipose tissue may be involved in regulating fetal growth and development and it may be related to energy intake, storage and expenditure. In preterm male infants serum leptin concentration increases with postnatal weight and testosterone may suppress leptin synthesis.

The EMG Development of the Longissimus and Multifidus Muscles After Plugging the Horizontal Semicircular Canals

The effects of plugging both horizontal semicircular canals at the 5th postnatal day (P5) on the development of the EMG of two back muscles (the M. longissimus lateralis and the M. multifidus) were studied in rats between P5 and P46. The EMG was related to locomotion, grooming, and rearing of these rats. Special attention was paid to locomotor behavior. After this vestibular deprivation, the development of a consistent temporal relation between burst activity in the EMG of both Mm. longissimii laterales and the swing phase of one hind paw was retarded by about 3 days. The development of coupling of maximal activity in the M. longissimus lateralis to the swing phase of one of the hind paws was even more retarded. No differences were observed between normal and vestibularly deprived rats during the development of activity in the Mm. multifidi. Our results indicate that information from the horizontal semicircular canals is essential for dynamic postural development and probably, therefore, for motor development in general. Furthermore, lack of such information has effects on the fine tuning of back muscle activity during locomotion at least until P40.

Functional and structural properties of Na+/K+‐ATPase enzyme in neonatal erythrocytes

The Na+/K(+)-pump is the main regulator enzyme of intracellular monovalent cation concentration. There are only limited data available concerning its structure and function in healthy neonates, in comparison with data available regarding its structure and function in children. Samples of 100 microL of anticoagulated blood were taken from 53 healthy neonates (age under 6th postnatal day, median age 3.5 days) and 61 healthy children (median age 12.4 months, range 6-36 months,). The Na+/K(+)-ATPase activity, its sensitivity to ouabain (a digoxin-analogue substance) and the expression of Na+/K(+) ATPase subunit isoforms were determined. The enzyme activity (429.2 +/- 17.2 versus 295.5 +/- 10.2 U, P < 0 x 0.001) and I50 value for ouabain inhibition (1.50 +/- 0.10 versus 0.96 +/- 0.10 mumol L-1, P < 0.05) was higher in neonates. More alpha 1 subunits (relative density: 1.16 +/- 0.10 versus 0.75 +/- 0.03, P < 0.001) and higher alpha 1/ alpha 2 ratio (4.14 +/- 0.21 versus 2.02 +/- 0.16, P < 0.01) were detected. This is the first study demonstrating changes of Na+/K(+) -ATPase molecules not only in enzyme activity, but also on protein level. Our results might contribute to the understanding of the resistance of neonatal cell membranes toward the pharmacodynamic actions of cardiac glycosides.

Motor Development after Vestibular Deprivation in Rats

GEISLER, H.C. AND A. GRAMSBERGEN. Motor development after vestibular deprivation in rats. NEUROSCI BIOBEHAV REV 22(4) 565–569, 1998.—This review summarizes the postural development in the rat and the influences of vestibular deprivation from the 5th postnatal day on this development. Vestibular deprivation leads to a delay in motor development. Most probably this delay is caused by a delay in the development of postural control, which is characterized by a retarded EMG development in postural muscles. Our results indicate that the developing nervous system cannot compensate for a vestibular deficit during the early phase of ontogeny.

Spontaneous congenital hydrocephalus in the mutant mouse hyh. Changes in the ventricular system and the subcommissural organ.

The subcommissural organ is an ependymal gland located at the entrance of the cerebral aqueduct. It secretes glycoproteins into the cerebrospinal fluid, where they aggregate to form Reissner's fiber. This fiber grows along the aqueduct, fourth ventricle, and central canal. There is evidence that the subcommissural organ is involved in the pathogenesis of congenital hydrocephalus. This organ was investigated in the mutant mouse hyh developing a congenital hydrocephalus. The central nervous system of normal and hydrocephalic hyh mice, 1 to 40 days old, was investigated using antibodies recognizing the subcommissural organ secretory glycoproteins, and by transmission and scanning electron microscopy. At birth, the affected mice displayed open communications between all ventricles, absence of a central canal in the spinal cord, ependymal denudation of the ventricles, stenosis of the rostral end of the aqueduct, and hydrocephalus of the lateral and third ventricles and of the caudal end of the aqueduct. Around the 5th postnatal day, the communication between the caudal aqueduct and fourth ventricle sealed, and hydrocephalus became severe. It is postulated that the hyh mice carry a genetic defect affecting the ependymal cell lineage. The subcommissural organ showed signs of increased secretory activity; it released to the stenosed aqueduct a material that aggregated, but it did not form a Reissner's fiber. A large area of the third ventricular wall differentiated into a secretory ependyma synthesizing a material similar to that secreted by the subcommissural organ. It is concluded that the subcommissural organ changes during hydrocephalus; whether these changes precede hydrocephalus needs to be investigated.

The Effects of Prenatally Administered Endogenous Cannabinoid on Rat Offspring

We reported previously that the main psychoactive component of marihuana, Δ 9 tetrahydrocannabinol (THC), when injected prenatally, temporarily inhibited the developing hypothalamo–pituitary system in rat offspring. In the present study, we investigated the effects of the recently described endogenous ligand for the central cannabinoid receptor, arachidonyl-ethanolamide (anandamide, ANA), on the postnatal development of the hypothalamo–pituitary axis (HPA) when administered during the third week of gestation. Rat pups were killed every fifth day from delivery to the 20th postnatal day; gonads, pituitary, and rest of body were weighed, and samples were collected for analysis of gonadotropin releasing hormone in the hypothalamus and luteinizing hormone, follicle stimulating hormone, prolactin, and growth hormone in the pituitaries and sera. The effects of ANA and THC were compared. Both ANA and THC caused predominantly inhibitory effects on the measured parameters. The inhibition was most pronounced immediately following delivery, whereas at the end of the investigated period (20th postnatal day) no differences were observed. We conclude that endogenous and exogenous cannabinoids have similar but slightly different effects on the developing HPA and that the action is transitory. We postulate that ANA probably acts via central cannabinoid receptors and/or neuroendocrine receptors to function as a neuromodulator.

The effect of starvation on brain carnitine concentration in neonatal rats.

We examined carnitine concentrations in fasted neonatal rat brain to evaluate the effect of starvation on fatty acid metabolism. The free- and acylcarnitine concentrations in neonatal rat brain and heart were determined after a 72-hour starvation period from the 3rd to 6th postnatal day. They were also determined in rats at 3 and 6 days of age fed normally by the mother rats as controls. In the brain, the mean free carnitine concentration in the fasted group showed an increase similar to that in normal rats and there was no difference between the fasted and 6-day-old control rats. However, the mean acylcarnitine concentration was significantly higher in the fasted group than in the control group at both 3 and 6 days of age. Almost all of the increased acylcarnitine in the fasted group was short-chain acylcarnitine. In the heart, there was no difference in the mean free carnitine concentration between the fasted group and control group at 6 days of age. The 6-day-old rats in both the fasted and control groups showed higher levels compared to 3-day-old rats in the control group. The mean acylcarnitine concentration in the fasted group was not different from that in control group at 6 days of age, while the amount of short-chain acylcarnitine was less than that in the control group at 6 days of age. These findings suggest that in the brain, carnitine is accumulated as a result of redistribution during starvation, and is utilized for energy supply by fatty acid oxidation.

The effects of early vestibular deprivation on the motor development in the rat

The motor development after uni- or bilateral vestibular deprivation from the 5th or 16th postnatal day was studied in 23 rats between the 6th and the 60th day. Their motor behaviour was compared to that of 17 normal rats. Vestibular deprivation from the 5th day causes a marked retardation in motor development. Our results suggest that this retardation is caused by non-optimal stabilization of the trunk during development. Vestibular deprivation from the 16th day causes only short lasting disturbances of motor behaviour for one or two days. The main permanent effect of bilateral vestibular deprivation is head oscillation during locomotion. The temporal organization of the motor behaviour of rats bilaterally deprived from the 5th day was disturbed as well. These rats show more bouts of locomotion, rearing and pivoting.

Development of 11β -Hydroxysteroid Dehydrogenase Expression in the Rat Cochlea

11β-hydroxysteroid dehydrogenase (11β-HSD) catalyzes the transformation of active glucocorticoid (GC) steroids to inactive 11-oxosteroids, as well as the reverse reaction. 11β-HSD was previously demonstrated specifically in the spiral ligament of the lateral cochlear wall where it was co-localized with adrenal steroid receptors. These findings imply that 11 β-HSD regulates binding of corticoids to their inner ear receptors. The GC receptor expression initially occurs around the critical maturation period of the cochlear duct. 11 β-HSD, which is an integral part of the cochlear steroid receptor system, could indirectly affect glucocorticoid-mediated induction processes. In this study the expression of 11β-HSD was studied in the postnatal rat cochlea from the 3rd to 30th postnatal day. Bouin's fixed, paraffin-embedded cochlear sections were processed for immunocytochemical detection of 11β-HSD using polyclonal antibodies against 11 β-HSD. 11β-HSD expression appeared at the 12th postnatal day at low levels in spiral ligament tissues. From the 15th postnatal day, 11β-HSD expression was stronger and similar to that of the adult cochlea. No additional inner ear tissue region expressed 11 β-HSD enzyme during the observed period. 11β-HSD expression coincides with the onset of functional maturity of the rat cochlear duct. The expression of 11β-HSD is preceded by the expression of GC receptors which appeared at the 7th postnatal day in the rat cochlea. These results further suggest an integrative role of the cochlear steroid receptor system in the homeostasis and functional maturation of the cochlea.

Functional properties of receptors for glutamate and GABA in cultured trigeminal ganglion neurons

Properties of receptors for glutamate, consisting of kainate, alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA), and gamma-aminobutyric acid (GABA) in the rat trigeminal ganglion (TG) neurons were studied by means of the whole-cell patch-clamp technique. 1. All TG neurons (diameter 20 approximately 40 microns) responded to GABA, but none of them responded to AMPA, NMDA, and glycine. TG neurons responding to kainate were smaller than 25 microns and desensitized by prolonged agonist exposure. 2. On the basis of the dose-response curves, EC50 and Hill coefficient were found to be 13. 7 microM and 1.2 for kainate, while those for GABA were 28.6 microM and 2.0, respectively. 3. Most kainate receptors in the TG neurons showed linear current-voltage (I-V) relations, though some showed inward rectification. The "Instantaneous" I-V relationship for GABA was linear, where as the "steady-state" I-V relationship produced by ramp changes in potential showed outward rectification in some neurons. 4. The number of TG neurons responding to kainate increased until the 8th postnatal day, while all the recorded neurons responded to GABA in postnatal days. The results indicated that there are kainate and GABAA receptors in the TG neurons and suggested that they are composed of various subunits.

Tectorial membrane-organ of Corti relationship during cochlear development.

The development of stereociliary attachment to the tectorial membrane was investigated in the mouse cochlea using transmission and scanning electron microscopy. At the 18th gestational day, only the major tectorial membrane can be identified covering the greater epithelial ridge and the inner hair cells in all turns. At the 19th gestational day, the minor tectorial membrane was first seen in the basal turn, over the outer hair cells. During early stages of development, the stereocilia of hair cells were surrounded by a loose fibrillar material underneath the tectorial membrane. After the 10th postnatal day, the outer hair cells' stereocilia were attached to Kimura's (or Hardesty's) membrane, while inner hair cells' stereociliary bundles were attached to the undersurface of the tectorial membrane near the Hensen's stripe. Between the 10th and the 14th postnatal days, the space between the inner hair cells and the first row of outer hair cells widened by virtue of the growth of the heads of pillar cells, and the inner hair cells' stereocilia were displaced towards the Hensen's stripe. After the 14th postnatal day, the inner hair cells' stereociliary bundles detached from the tectorial membrane, while the outer hair cells' stereocilia remained attached to it. The tip-link system, which connects the tips of the stereocilia to the next tallest stereocilia, is present at birth in the outer hair cells. The marginal pillar, that anchored the tectorial membrane to the underlying organ of Corti during development, first appeared on the 6th postnatal day and disappeared on the 14th-15th postnatal day. The present data together with other reports support the idea that although some structures, such as hair cells' stereocilia and innervation, are already formed early during development, the cochlear microarchitecture is not fully developed morphologically and ready to function normally until the end of the second postnatal week in the mouse.