Growth In Neonatal Rats Research Articles

Co-Enzyme Q10 and n-3 Polyunsaturated Fatty Acid Supplementation Reverse Intermittent Hypoxia-Induced Growth Restriction and Improved Antioxidant Profiles in Neonatal Rats.

Neonatal intermittent hypoxia (IH) increases the risk for many morbidities in extremely low birth weight/gestational age (ELBW/ELGA) neonates with compromised antioxidant systems and poor growth. We hypothesized that supplementation with coenzyme Q10 (CoQ10, ubiquinol) or n-3 polyunsaturated fatty acids (PUFAs) during neonatal IH improves antioxidant profiles and somatic growth in neonatal rats. Newborn rats were exposed to two IH paradigms at birth (P0): (1) 50% O2 with brief hypoxic episodes (12% O2); or (2) room air (RA) with brief hypoxia, until P14 during which they received daily oral CoQ10 in olive oil, n-3 PUFAs in fish oil, or olive oil only from P0 to P14. Pups were studied at P14 or placed in RA until P21 for recovery from IH (IHR). Body weight and length; organ weights; and serum antioxidants and growth factors were determined at P14 and P21. Neonatal IH resulted in sustained reductions in somatic growth, an effect that was reversed with n-3 PUFAs. Improved growth was associated with higher serum growth factors. CoQ10 decreased superoxide dismutase (SOD) and glutathione, but increased catalase, suggesting reduced oxidative stress. Further studies are needed to determine the synergistic effects of CoQ10 and n-3 PUFA co-administration for the prevention of IH-induced oxidative stress and postnatal growth deficits.

Ontogenic expression of key ADME genes and their roles in imatinib‐induced toxicity in juvenile rats

Imatinib is an oral tyrosine kinase inhibitor that is approved for the treatment of pediatric chronic myeloid leukemia. Prepubertal growth impairment has been reported to be a major neurologic adverse effect of long‐term imatinib treatment in children. Numerous studies have demonstrated that cytochrome P450 3A4 and ABCB1 are major ADME genes that play critical roles in biotransformation and brain disposition of imatnib. In the present study, we first evaluated the relationships between ontogentic expression of ADME genes and exposure of imatinib in a neonatal rat model. The expression of Cyp3a1 and Abcb1 of rats from different postpartum days (PPD) were determined at mRNA and protein levels, and results revealed that their expression increased with age. Pharmacokinetic studies after single oral dose of imatinib (30 mg/kg) given to 7‐, 14‐, 21‐, 28‐ or 35‐day‐old rats demonstrated that brain distribution of imatinib decreased with age, with > 20 fold decrease in PPD35 rats compared with PPD7 rats, consistent with mRNA and protein expression levels of key ADME genes. Juvenile rat toxicity studies with oral imatinib were conducted to examine the relationship between Cyp3a1 and Abcb1 gene expression and growth (in particular bone formation) retardation. Preliminary data suggest a distinctive inhibitory effect of imatinib on bone growth in neonatal rats. Mechanistic investigations using osteoblasts and in vivo neonatal rat model are on‐going to delineate regulatory network between imatinib disposition and bone formation.Support or Funding InformationThe current work was supported in part by a grant from Soochow University, Suzhou, China [Grant No. Q413200711], and a grant from the National Science Foundation of China [Grant No. 81473278].

Short-term treatment with VEGF receptor inhibitors induces retinopathy of prematurity-like abnormal vascular growth in neonatal rats

Retinal arterial tortuosity and venous dilation are hallmarks of plus disease, which is a severe form of retinopathy of prematurity (ROP). In this study, we examined whether short-term interruption of vascular endothelial growth factor (VEGF) signals leads to the formation of severe ROP-like abnormal retinal blood vessels. Neonatal rats were treated subcutaneously with the VEGF receptor (VEGFR) tyrosine kinase inhibitors, KRN633 (1, 5, or 10 mg/kg) or axitinib (10 mg/kg), on postnatal day (P) 7 and P8. The retinal vasculatures were examined on P9, P14, or P21 in retinal whole-mounts stained with an endothelial cell marker. Prevention of vascular growth and regression of some preformed capillaries were observed on P9 in retinas of rats treated with KRN633. However, on P14 and P21, density of capillaries, tortuosity index of arterioles, and diameter of veins significantly increased in KRN633-treated rats, compared to vehicle (0.5% methylcellulose)-treated animals. Similar observations were made with axitinib-treated rats. Expressions of VEGF and VEGFR-2 were enhanced on P14 in KRN633-treated rat retinas. The second round of KRN633 treatment on P11 and P12 completely blocked abnormal retinal vascular growth on P14, but thereafter induced ROP-like abnormal retinal blood vessels by P21. These results suggest that an interruption of normal retinal vascular development in neonatal rats as a result of short-term VEGFR inhibition causes severe ROP-like abnormal retinal vascular growth in a VEGF-dependent manner. Rats treated postnatally with VEGFR inhibitors could serve as an animal model for studying the mechanisms underlying the development of plus disease.

Effect of gender on long‐term effects of catch‐up growth in neonatal rats

Poor early growth in preterm infants leads to impaired neurodevelopment but may reduce the risk of metabolic syndrome. Although elevated leptin levels in young animals may increase the risk of later leptin resistance, hepatic steatosis and metabolic syndrome, it is required for normal neurocognitive development. We therefore examined the effect of differences in early growth rates on serum leptin, triglycerides (TG) and cognition. Newborn rat pups were randomized into normal (N, n=10) or reduced growth (R, n=16) litters. On d11, R pups were re‐randomized into litters creating catch‐up (R‐6, n=6), normal (R‐10, n=10) or reduced growth (R‐16, n=10). Serum leptin, serum TG and hepatic TG were measured at d 10, 22, and 60; cognition in a T‐maze on d35. Leptin was higher in N pups than R on d10 (p<0.0001), and N animals scored highest on the T‐maze. In females, leptin on d22 and T‐maze score were significantly lower in the R‐16 group than other R groups. On d10, serum TG was significantly higher in N than R pups (p=0.001). In females, hepatic TG was significantly greater in R‐6 and N animals than in R‐10 or R‐16. We conclude that early growth restriction leads to lower serum leptin, serum TG, and impaired long‐term cognitive development. Subsequent catch‐up growth improves cognition, but worsens hepatic steatosis, in female rats. This model may contribute to our understanding of poor developmental outcomes in preterm infants.Grant Funding Source: UC Davis Department of Pediatrics

Tert-Butyl hydroperoxide, an organic peroxide, causes temporary delay in hair growth in a neonatal rat model

tert-butyl hydroperoxide (tBHP), an organic peroxide, has been shown to cause irreversible damage to keratinocytes in vitro with prolonged administration at high concentrations, and reversible damage with short-term administration at low concentrations. To investigate the effects of tBHP on keratinocytes in vivo, we analysed hair growth in tBHP-treated neonatal rats. Sprague-Dawley and Long-Evans rat pups were injected subcutaneously with tBHP or vehicle once daily for 6 days, and hair growth was monitored. The tBHP-treated rats had a significant delay in hair growth. However, this delay reversed within days, and the hair coats, including hair pigmentation, of tBHP-treated and sham-treated rats were indistinguishable 2 weeks later. Histological analysis and BrdU labelling of S phase cells confirmed the delay in hair-follicle growth and its reversal in tBHP-treated rats. Our results indicated that the changes incurred in hair follicles by short-term use of high-dose oxidants in vivo are temporary and reversible.

Effect of increased pressure loading on heart growth in neonatal rats

During embryonic and fetal development, the ventricular myocardium increases its mass principally by adding new cells (hyperplasia), while postnatally, it does so mainly through increase of cell size (hypertrophy). Switching between these two mechanisms of adaptation to increasing functional demand occurs in the early neonatal period. We investigated the response of the neonatal rat left ventricle to pressure overload induced by constriction of the abdominal aorta at postnatal day 2. Sampling for morphological examination with measurements of ventricular wall thickness and myocyte width was performed at days 2, 3, 5, 10, and 21. 3H-thymidine pre-labeling with label dilution was used to assess proliferative history at day 21, and bromodeoxyuridine labeling was used to measure the rates of DNA synthesis at each time point. The left ventricular wall was significantly thicker than in controls in the AC group from day 3, while thickness of individual myocytes was not increased until day 10. Label dilution showed evidence of higher number of cellular divisions correlating with severity of the phenotype in the AC group at day 21. Terminal DNA synthesis index was increased significantly at day 3, but there was no significant difference from controls at days 5, 10, or 21. Apoptotic rates were not different from controls at any sampling interval. Together, these results suggest that adaptation of the neonatal myocardium to increased pressure load is rapid, and is based on transitory hyperplasia followed by hypertrophy of myocytes.

Effect of the 21-aminosteroid U74389G on oxygen-induced free radical production, lipid peroxidation, and inhibition of lung growth in neonatal rats.

Bronchopulmonary dysplasia is a chronic pneumopathy of preterm infants, with significant associated mortality and morbidity, for which there is no effective preventive therapy. Pulmonary O2 toxicity is thought to be a major contributor to the development of bronchopulmonary dysplasia, and antioxidant interventions hold significant promise for therapy. The relative importance of specific reactive oxygen species in the development of O2-mediated lung injury is unknown. In this study, we tested the effect of a synthetic 21-aminosteroid, U74389G, on 95% O2-induced free radical production, lipid peroxidation, and inhibition of postnatal lung growth in a neonatal rat model. Lipid peroxidation products, as measured by total 8-isoprostane and aldehydes, and hydroxyl radical formation, assessed using salicylate metabolites, in rat lungs and serum were significantly increased after exposure to 95% O2. These changes could be completely or partially attenuated by U74389G. However, U74389G did not improve the survival rate or lung wet-to-dry weight ratio. Expression of proliferating cell nuclear antigen, a marker for DNA synthesis, was examined by immunohistochemistry. Four- or 7-d-old control rat lungs had active DNA synthesis, which was inhibited by exposure to 95% O2. U74389G had a protective effect against 95% O2-mediated inhibition of DNA synthesis. Air-exposed animals treated with U74389G had a modest reduction in lung DNA synthesis, consistent with a role for hydroxyl radicals or lipid hydroperoxides as second messengers in the normal regulation of lung growth.

Effect of the 21-Aminosteroid U74389G on Oxygen-Induced Free Radical Production, Lipid Peroxidation and Inhibition of Lung Growth in Neonatal Rats

Effect of the 21-Aminosteroid U74389G on Oxygen-Induced Free Radical Production, Lipid Peroxidation and Inhibition of Lung Growth in Neonatal Rats

Protective effect of ginseng saponins against impaired brain growth in neonatal rats exposed to ethanol.

This study was performed to determine the active constituents of the root of Panax ginseng C. A. Mayer in the amelioration of ethanol-induced impediment of brain growth in the neonatal stage. To establish an animal model of the brain growth impediment caused by ethanol, ethanol (6 g/kg s.c.) was administered to rat pups on postnatal day 6, which corresponded to the third trimester of pregnancy for humans. Brain weight, especially cerebellar weight, was significantly reduced in the ethanol-exposed pups. In contrast, neither separation from dams nor pentobarbital treatment affected brain weight. A saponin fraction of ginseng extract prevented this ethanol-induced reduction of brain weight. Some ginseng saponins including ginsenosides Rg1, Rb2, Rd, Rf and Re effected stimulated a potent recovery of cerebellum growth in this animal model.

Effects of continuous infusion of insulin-like growth factor I and II, alone and in combination with thyroxine or growth hormone, on the neonatal hypophysectomized rat

In this study, we examined the effects of systemically administered insulin-like growth factor (IGF)-I and -II on growth of the hypophysectomized (Hx) neonatal rat. Neonatal Wistar rats were Hx or sham Hx on postnatal day (PND) 6 and implanted sc with Alzet pumps on PND 10. Recombinant human IGF-I or -II were infused between PND 10 and 18 at an average dose of 1.9 micrograms/g body weight (BW) per day. In addition, some groups received daily sc injections of recombinant human GH or thyroxine (T4) at 2.5 micrograms and 25 ng/g BW per day, respectively. Pups were sacrificed on PND 18 and serum IGF levels determined. Despite restoration of serum IGF-I levels to sham control values in the Hx pups infused with IGF-I, no significant increase in BW occurred, although some increase in individual organ growth was observed (spleen, kidney, lung). Similarly, administration of IGF-II proved ineffective as a growth promoter in the neonatal Hx rat. In contrast, GH alone stimulated BW gain (P less than 0.001). T4 proved most potent in increasing skeletal growth (50% increase over Hx controls, P less than 0.001), without increasing serum IGF-I or -II levels. IGF-I and GH were equally effective in promoting a small yet statistically significant (17% over Hx controls, P less than 0.05) increase in skeletal growth. A synergistic effect on BW was observed with combined administration of T4 plus IGF-I to the Hx pups (P less than 0.05). The effects of hormonal therapy on serum IGF binding proteins (IGFBPs) was assessed by Western ligand blots. Administration of IGF-I, but not GH, resulted in increased levels of IGFBP-3, the predominant IGFBP of the adult rat. We conclude that systemically administered IGFs in doses that result in normalization of serum levels are ineffective promoters of somatic growth in neonatal rats. While normalization of serum IGF-I levels does result in modest skeletal growth, selective organ growth and increased serum IGFBP-3, growth stimulation does not equal that seen with GH (body weight) or thyroid hormone (skeletal growth). Differences in IGFBP profiles fail to account for the increased potency of GH as a promoter of BW gain. Thus, our data do not support a major endocrine role for IGF-I or -II in neonatal growth, but are consistent with an autocrine/paracrine action of IGF in the mediation of neonatal mammalian growth.

Effects of continuous infusion of insulin-like growth factor I and II, alone and in combination with thyroxine or growth hormone, on the neonatal hypophysectomized rat.

In this study, we examined the effects of systemically administered insulin-like growth factor (IGF)-I and -II on growth of the hypophysectomized (Hx) neonatal rat. Neonatal Wistar rats were Hx or sham Hx on postnatal day (PND) 6 and implanted sc with Alzet pumps on PND 10. Recombinant human IGF-I or -II were infused between PND 10 and 18 at an average dose of 1.9 micrograms/g body weight (BW) per day. In addition, some groups received daily sc injections of recombinant human GH or thyroxine (T4) at 2.5 micrograms and 25 ng/g BW per day, respectively. Pups were sacrificed on PND 18 and serum IGF levels determined. Despite restoration of serum IGF-I levels to sham control values in the Hx pups infused with IGF-I, no significant increase in BW occurred, although some increase in individual organ growth was observed (spleen, kidney, lung). Similarly, administration of IGF-II proved ineffective as a growth promoter in the neonatal Hx rat. In contrast, GH alone stimulated BW gain (P less than 0.001). T4 proved most potent in increasing skeletal growth (50% increase over Hx controls, P less than 0.001), without increasing serum IGF-I or -II levels. IGF-I and GH were equally effective in promoting a small yet statistically significant (17% over Hx controls, P less than 0.05) increase in skeletal growth. A synergistic effect on BW was observed with combined administration of T4 plus IGF-I to the Hx pups (P less than 0.05). The effects of hormonal therapy on serum IGF binding proteins (IGFBPs) was assessed by Western ligand blots. Administration of IGF-I, but not GH, resulted in increased levels of IGFBP-3, the predominant IGFBP of the adult rat. We conclude that systemically administered IGFs in doses that result in normalization of serum levels are ineffective promoters of somatic growth in neonatal rats. While normalization of serum IGF-I levels does result in modest skeletal growth, selective organ growth and increased serum IGFBP-3, growth stimulation does not equal that seen with GH (body weight) or thyroid hormone (skeletal growth). Differences in IGFBP profiles fail to account for the increased potency of GH as a promoter of BW gain. Thus, our data do not support a major endocrine role for IGF-I or -II in neonatal growth, but are consistent with an autocrine/paracrine action of IGF in the mediation of neonatal mammalian growth.

Enhancement of intestinal growth in neonatal rats by epidermal growth factor in milk.

Breast milk has been shown to enhance neonatal intestinal growth. Because epidermal growth factor (EGF) is present in the milk of various mammalian species, the hypothesis was tested that EGF in rodent milk mediates, in part, the breast milk-enhanced intestinal growth in neonatal rat. Fifty-eight rat pups fed artificial formula that contained 1.2, 3.0, and 6.0 micrograms/ml EGF for 39 h had greater incorporation of [3H]thymidine into DNA and DNA content of intestine than 29 pups fed unsupplemented formula. Pups fed EGF for 5 days had significantly greater body weight, intestinal weight, length, and DNA content than control pups. Conversely, pups fed pooled rat milk containing rabbit-derived antibody to EGF for 39 h had intestines of lower weight that contained less DNA than animals fed rat milk containing normal rabbit serum. EGF appears to mediate in part, breast milk-enhanced neonatal intestinal growth.

Breast-Milk-Enhanced Intestinal and Somatic Growth in Neonatal Rats

Ten rat pups fed pooled rat milk and 9 pups fed artificial formula had significantly heavier stomachs and intestines that contained more DNA and RNA by 40 h of age than 12 pups sacrificed at birth. Breast-milk-fed animals had significantly heavier intestines, livers and kidneys than animals fed artificial formula. The intestines of pups fed breast milk contained more DNA and RNA with a lower RNA:DNA ratio than the intestines of pups fed artificial formula. Hence, when caloric intake and mode of feeding are controlled, natural milk enhances neonatal intestinal growth more than artificial formula.

Blood alcohol concentration: A critical factor for producing fetal alcohol effects

A dose of 6.6 g/kg of alcohol, delivered in 12 equally-spaced fractions each 24 hours via an artificial rearing procedure during postnatal days 4–10, produced mean blood alcohol concentrations (BACs) that were low (46.6 mg/dl), but stable with time. This relatively constant alcohol exposure did not limit brain growth in neonatal rats when measured on postnatal day 10. The same daily dose concentrated into 6 fractions over 12 hours (with 6 alcohol-free fractions the remaining 12 hours) resulted in cyclic BACs having high peaks (270.2 mg/dl). The cyclic regimen produced a significant reduction in brain growth. Thus, the peak blood alcohol concentration is a critical factor in determining the minimum dose for producing microencephaly and must be considered when estimating the relative teratogenic risks of alcohol intake during pregnancy.

Use of isolated myocytes to determine hyperplastic or hypertrophic myocardial cell growth in neonatal rats and turkeys

Use of isolated myocytes to determine hyperplastic or hypertrophic myocardial cell growth in neonatal rats and turkeys

Impaired brain growth in neonatal rats exposed to ethanol.

Infant rat pups, fed through intragastric cannulas from postnatal day 4 through day 18, showed a 19 percent reduction in total brain weight when ethanol was included in their diet on days 4 through 7. This reduction in brain weight occurred even though body growth in the experimental rats was equal to that of their littermate controls. The ethanol-exposed animals were markedly hypoactive during the period of drug administration, then displayed gross body tremors for 3 to 5 days. Throughout the study, the animals treated with ethanol had poor motor coordination and were hyperresponsive. These brain and behavioral effects appear similar to those seen in fetal alcohol syndrome.

Effects of hypoxia on cardiac growth in neonatal rat.

To determine whether low oxygen environments enhance cardiac cell division in the neonatal period, newborn rat pups were reared for 21 days in 12-15% oxygen. Left ventricle and right ventricle weights were 30 and 180% greater than controls matched for body weight (P less than 0.001) as were left ventricle/body weight ratios (3.68+/-0.26 vs. 2.99+/-0.05 mg LV/g body wt,P less than 0.001). Left ventricular total DNA and DNA concentration was 95 and 48% greater than controls (P less than 0.001). Autoradiography confirmed that this increase in ventricular DNA resulted from an increased rate of division of cardiac muscle cells, fibroblast, and vascular endothelial cells. When [3H]thymidine was injected on day ), autoradiographs prepared on day 21 reflected an increased dilution of label in hypoxic rats consistent with enhanced proliferation. The labeling index and grains per nucleus of ventricular muscle cells was 25% (P less than 0.01) and 20% (P less than 0.02) less than controls, Thus, hypoxic stress applied early in the neonatal period augments the rate of division and ultimate number of cardiac muscle cells. Whether this enhancement results from a primary effect of oxygen or from secondary hemodynamic factors remains unknown.

Chronic Isoproterenol Treatment of Neonatal Rats

The chronic subcutaneous administration of isoproterenol to neonatal and young male and female rats resulted in statistically significant reductions of the norepinephrine concentrations of the heart and submaxillary and parotid glands and increased concentrations in the stellate ganglion. However, the total norepinephrine content of these tissues was not affected. The epinephrine levels of these tissues were not significantly different from controls except for an increased total content in the submaxillary glands. Chronic treatment did not alter the norepinephrine levels of the brain, superior cervical ganglion, adrenal glands, vas de-ferens, uterus, spleen, kidneys, lungs, and small intestine. Growth of neonatal rats was retarded. The wet weights of the submaxillary and parotid glands were significantly greater, while the spleen, uterus, adrenal glands, and stellate ganglion weighed less in iso-proterenol-treated than in control rats. The wet weights of other peripheral tissues were not affected. The ratios of the tissue weight to the body weight were significantly increased for the heart and submaxillary and parotid glands and decreased for the uterus. The first dose of isoproterenol produced hypothermia in mature rats but hyperthermia in newborn rats. Sparse hair growth in neonatal rats and hair loss in weanling rats were also observed during chronic treatment.