P3 Animals Research Articles

P13.16 Metastatic potential of systemic glioblastoma stem cell lines in vivo

Abstract BACKGROUND Despite aggressive tumor behavior, extracranial metastases rarely develop in glioblastoma (GBM) patients. Two potential explanations have been suggested: 1) The blood-brain-barrier functions as a physical barrier that prevents the dissemination of GBM cells out of the central nervous system (CNS) or 2) that extracranial metastasis do occur, but the patients die before extracranial metastases manifest themselves. The first theory has been questioned based on the fact that circulating tumor cells (CTC) were found in blood samples of GBM patients without systemic metastases. To date it has not been proven if CTCs are able to reenter the brain and to what extent they are able to form systemic extracranial metastatic lesions. Therefore, the current study aimed at analyzing the dissemination patterns and the underlying mechanisms associated with the ability of GBM CTCs to form extracranial metastases. MATERIAL AND METHODS Five highly characterized human GBM stem cell (GSC) lines (P3, BG5, BG7, GG6, GG16), displaying GBM CNV patterns, were intracranially implanted in a first cohort, then transduced with a lentiviral Firefly Luciferase-eGFP vector and injected into the left cardiac ventricle of NOD/SCID mice in a second cohort. Mice were observed closely and tumor burden was assessed using in vivo as well as ex vivo bioluminescence imaging, MRI and PET. Mice were euthanized when the objective endpoint criteria (tumor burden) was met, then organs were harvested and fixed for further analysis. RESULTS First, a detailed characterization of the GSC line invasion patterns were assessed when grown as orthotopic xenografts in vivo dividing them into three categories: 1) Highly invasive without apparent angiogenesis (BG5) 2) Invasive with perivascular infiltration and angiogenesis (P3, BG7 and GG16) and 3) Angiogenic and highly circumscribed (GG6). Following intracardial injection, (7 out of 8) P3 animals developed extracranial and intracranial tumors with a distinctive pattern. Brain, adrenal gland, ovary and liver were amongst the organs most susceptible for tumor growth in the P3 group. For the BG5 and BG7 cell lines, no metastases were observed whereas only 1 animal out of 10 developed metastases in both groups GG16 and GG6. CONCLUSION Only one out of 5 GSC lines exhibited a strong metastatic potential when injected into the left cardiac ventricle. Compared to other tumors which exhibit a strong metastatic potential from the circulation, GSC lines do only to a very limited extent show this potential reflecting observations made in the clinic.

Differential glucose and beta-hydroxybutyrate metabolism confers an intrinsic neuroprotection to the immature brain in a rat model of neonatal hypoxia ischemia

Neonatal hypoxia ischemia (HI) is the main cause of newborn mortality and morbidity. Preclinical studies have shown that the immature rat brain is more resilient to HI injury, suggesting innate mechanisms of neuroprotection. During neonatal period brain metabolism experience changes that might greatly affect the outcome of HI injury. Therefore, the aim of the present study was to investigate how changes in brain metabolism interfere with HI outcome in different stages of CNS development. For this purpose, animals were divided into 6 groups: HIP3, HIP7 and HIP11 (HI performed at postnatal days 3, 7 and 11, respectively), and their respective shams. In vivo [18F]FDG micro positron emission tomography (microPET) imaging was performed 24 and 72 h after HI, as well as ex-vivo assessments of glucose and beta-hydroxybutyrate (BHB) oxidation. At adulthood behavioral tests and histology were performed. Behavioral and histological analysis showed greater impairments in HIP11 animals, while HIP3 rats were not affected. Changes in [18F]FDG metabolism were found only in the lesion area of HIP11, where a substantial hypometabolism was detected. Furthermore, [18F]FDG hypometabolism predicted impaired cognition and worst histological outcomes at adulthood. Finally, substrate oxidation assessments showed that glucose oxidation remained unaltered and higher level of BHB oxidation found in P3 animals, suggesting a more resilient metabolism. Overall, present results show [18F]FDG microPET predicts long-term injury outcome and suggests that higher BHB utilization is one of the mechanisms that confer the intrinsic neuroprotection to the immature brain and should be explored as a therapeutic target for treatment of HI.

Chemical disbudding of goat kids with subcutaneous administration of synthetic eugenol: Histopathology and morphometry.

This study was conducted to evaluate the effect of subcutaneous administration of synthetic eugenol (EG) for disbudding of goat kids, as a new chemical method. Thirty apparently healthy Raieni (Cashmere) goat kids (five-day-old) were divided randomly into six groups (n = 5). In the pathology (P) groups (P3, P8 and P60 according to the sample collection day after injection) an amount of 0.10 mL of EG was subcutaneously administrated in both horn buds. In the disbudding 1 and 2 (DB1, DB2) groups, 0.10 mL of EG, and in the control (C) group 0.10 mL normal saline was subcutaneously injected in the right horn buds, respectively. Eugenol injection in DB2 group was done in twelve-day-old goat kids. The left horn buds of DB1, DB2 and C groups were considered as control of horn outgrowing. The horn buds, kidneys, liver, lung, brain and heart, tissue specimens were collected from P3 and P8 groups, and bud skin samples were collected from P60 group. The results showed that the EG was able to stop the horn growth in the first week of goat life. There was no significant difference between left and right horn size in the C group. Histopathological study revealed complete necrosis of bud tissue in dermal and epidermal layers, in P3 animals. Healing and re-epithelialization were seen in the samples taken from P8 group. Subcutaneous injection of the synthetic EG can be considered as a new method for goat kids disbudding.

Impaired neurogenesis and associated gliosis in mouse brain with PEX13 deficiency

Zellweger syndrome (ZS), a neonatal lethal disorder arising from defective peroxisome biogenesis, features profound neuroanatomical abnormalities and brain dysfunction. Here we used mice with brain-restricted inactivation of the peroxisome biogenesis gene PEX13 to model the pathophysiological features of ZS, and determine the impact of peroxisome dysfunction on neurogenesis and cell maturation in ZS. In the embryonic and postnatal PEX13 mutant brain, we demonstrate key regions with altered brain anatomy, including enlarged lateral ventricles and aberrant cortical, hippocampal and hypothalamic organization. To characterize the underlying mechanisms, we show a significant reduction in proliferation, migration, differentiation, and maturation of neural progenitors in embryonic E12.5 through to P3 animals. An increasing reactive gliosis in the PEX13 mutant brain started at E14.5 in association with the pathology. Together with impaired neurogenesis and associated gliosis, our data demonstrate increased cell death contributing to the hallmark brain anatomy of ZS. We provide unique data where impaired neurogenesis and migration are shown as critical events underlying the neuropathology and altered brain function of mice with peroxisome deficiency.

PCR-SSCP Variation of IGF1 and PIT1 Genes and Their Association with Estimated Breeding Values of Growth Traits in Makooei Sheep

Molecular biology techniques genetic improvement by facilitating identification, mapping and analysis of polymorphism of genes by encoding proteins that act on metabolic pathways involved in economically interesting traits. This use of genetic markers can aid identification of those animals with the highest breeding values in sheep. On the basis of sheep genome mapping, information was examined on the ovine IGF1 and PIT1 genes as a possible genetic marker for growth traits in sheep. The current study was designed to estimate the frequencies of putative IGF-1 and PIT-1 genes SNPs and investigate associations with calculated EBVs of growth traits in Makooei sheep. PCR-SSCP analysis of the exon1 of IGF-I gene and include a part of intron2, exon3 and a part of intron3 and PIT-1 gene revealed the following banding patterns; three (AA, AG, GG) and four AA (p1), AB (p2), CC (p3), CD (p4), banding patterns respectively. Results from this study demonstrated higher performance of AA animals in BW and GBW, and AG animal in WW and W6 that may be related to the role of IGF-1 at the pre-puberty and puberty stages. Also higher performance of p3 animals in W9, YW and GSN, and p1 animal in GNY may be related to the PIT-1 role in post-puberty.

Delayed Post-Injury Administration of Riluzole Is Neuroprotective in a Preclinical Rodent Model of Cervical Spinal Cord Injury

Riluzole, a sodium/glutamate antagonist has shown promise as a neuroprotective agent. It is licensed for amyotrophic lateral sclerosis and is in clinical trial development for spinal cord injury (SCI). This study investigated the therapeutic time-window and pharmacokinetics of riluzole in a rodent model of cervical SCI. Rats were treated with riluzole (8 mg/kg) at 1 hour (P1) and 3 hours (P3) after injury or with vehicle. Afterward, P1 and P3 groups received riluzole (6 (mg/kg) every 12 hours for 7 days. Both P1 and P3 animals had significant improvements in locomotor recovery as measured by open field locomotion (BBB score, BBB subscore). Von Frey stimuli did not reveal an increase in at level or below level mechanical allodynia. Sensory-evoked potential recordings and quantification of axonal cytoskeleton demonstrated a riluzole-mediated improvement in axonal integrity and function. Histopathological and retrograde tracing studies demonstrated that delayed administration leads to tissue preservation and reduces apoptosis and inflammation. High performance liquid chromatography (HPLC) was undertaken to examine the pharmacokinetics of riluzole. Riluzole penetrates the spinal cord in 15 min, and SCI slowed elimination of riluzole from the spinal cord, resulting in a longer half-life and higher drug concentration in spinal cord and plasma. Initiation of riluzole treatment 1 and 3 hours post-SCI led to functional, histological, and molecular benefits. While extrapolation of post-injury time windows from rat to man is challenging, evidence from SCI-related biomarker studies would suggest that the post-injury time window is likely to be at least 12 hours in man.

Anesthetic Effects on the Developing Nervous System

Anesthetic Effects on the Developing Nervous System

Pretreatment with Low Doses of Erythropoietin Ameliorates Brain Damage in Periventricular Leukomalacia by Targeting Late Oligodendrocyte Progenitors: A Rat Model

Background: One of the pathological hallmarks of periventricular leukomalacia (PVL) is the selective vulnerability of late oligodendrocyte progenitors (preoligodendrocytes; preOLs) to hypoxia-ischemia (H-I). It is unknown whether recombinant human erythropoietin (rhEPO) protects preOLs in vivo. Objectives: To develop a rat PVL model in which preOLs are selectively damaged and exhibit similar pathological changes to diffuse-type human PVL, various conditions of H-I were compared in P2–P7 rats (P2 = postnatal day 2). To evaluate the effect of rhEPO on oligoprotection (preOLs), rhEPO was administered to P3 PVL rats. Methods: After counts of NG2-positive and O4-positive cells were performed in P2–P7 rats, right common carotid artery occlusion followed by 6% O₂ for 0–120 min was performed in P2–P4 rats. The mortality and histological alterations after hematoxylin/eosin staining and ED1 immunostaining were assessed 2 days after H-I. Various doses of rhEPO (1–30,000 U/kg i.p.) were administered to PVL rats 15 min before administration of 6% O₂. Results: Double-positive cells for NG2 and O4 were detected from P2, and their number gradually increased until P7. Although right common carotid artery occlusion with 6% O₂ for 60 min resulted in a relatively high proportion of deaths in P2–P4 rats, typical histological changes in the PVL diffuse component were found in most surviving P3 animals. With 50–100 U/kg rhEPO, the histological damage was attenuated. Conclusions: Histological changes similar to those seen in the PVL diffuse component were induced by H-I in P3 rats, in which preOLs were gradually developing, and a low dose of rhEPO was effective in the treatment of brain damage induced by H-I.

Characterisation of transverse slice culture preparations of postnatal rat spinal cord: preservation of defined neuronal populations

Spinal cord injury induces degenerative and regenerative processes and complex interactions of neurons with non-neuronal cells. In order to develop an in vitro tool for the investigation of such processes, we prepared and characterised spinal cord slice cultures (SCSC) from Wistar rats (p0-12). SCSC were sustained in vitro up to 12 days and characterised by immunohistochemistry. Calbindin+ neurons, distributed across the entire gray matter, were visible also after longer culture periods. NeuN+ neurons were best preserved in the dorsal horn whereas large NeuN+ and choline acetyltransferase+ motoneurons in the ventral horn vanished after 3 days in vitro. Nestin immunoreactivity was found in animals of all age groups, either in cells interspersed in the ependymal lining around the central canal or in cells resembling protoplasmic astrocytes. Glial fibrillary acidic protein+ astrocytes, initially restricted to the white matter, invaded the gray matter of SCSC early during the culture period. Microglial cells, stained by Griffonia simplicifolia isolectin B4, were rapidly activated in the dorsal tract and in the gray matter but declined in number with time. SCSC derived from p0 or p3 animals showed a better preservation of the cytoarchitecture than cultures derived from older animals. In summary, SCSC undergo degenerative changes, but they contain defined neuronal populations, the cytoarchitecture is partially preserved and the glial reaction is limited.

Assessment of factors regulating axon growth between the cortex and spinal cord in organotypic co-cultures: effects of age and neurotrophic factors.

Axon growth failure in the central nervous system (CNS) of adult animals is thought to be attributable to several factors, including an inadequate intrinsic growth response, the presence of inhibitory molecules, and a lack of adequate neurotrophic support. Here we use a new in vitro assay system to quantitatively assess growth of axons in cortex/spinal cord organotypic co-cultures from neonatal rats. Co-cultures of cortex and spinal cord were prepared from neonatal rats at P3 or P7, and by pairing cortex and spinal cords from different ages. Axon growth from the cortex to the spinal cord was assessed using DiI tract tracing techniques. Axons could be traced from the cortex to the spinal cord in co-cultures in which both tissues were obtained from P3 animals, whereas few axons crossed the cortex/spinal cord boundary in co-cultures from P7 animals. A larger number of axons could be traced across the boundary in co-cultures from P3 animals that were treated with neurotrophins (NGF, BDNF, or NT3), whereas neurotrophins produced minimal growth enhancement in P7 co-cultures. In mixed age co-cultures of P7 cortex with P3 spinal cord, moderate numbers of axons extended between the cortex and spinal cord when cultures were treated with neurotrophins, but few if any crossing axons were detected in co-cultures of P3 cortex with P7 spinal cords. These results indicate that successful growth of axons from the cortex to the spinal cord depends on the developmental age of the tissue terrain (the spinal cord and/or the interface between cortex and spinal cord explants), and to a lesser extent on the developmental state of the cortical neurons, and that axon growth between cortex and spinal cord can be enhanced by exogenous neurotrophins. These co-cultures provide a potentially useful assay for factors that affect axon growth that is intermediate between assays based on dissociated neurons and the intact tissue terrain.

Prenatal nicotine exposure increases apnoea and attenuates nicotinic potentiation of hypoglossal output in mice

Maternal smoking is associated with an increased risk of Sudden Infant Death Syndrome (SIDS) and nicotine is implicated as a causative agent due to adverse effects on central nervous system development [1]. The consequences of these changes for respiration are uncertain, but there is growing consensus that altered respiratory control contributes to the reduced ability of nicotine-exposed animals to tolerate hypoxia [2,3]. Most studies have focussed on overall changes in ventilation, but paid minimal attention to breathing pattern. In addition, responses have rarely been examined in the earliest neonatal periods when immature control mechanisms are more likely to contribute to unstable breathing, frequent apnoeas and hypoxic episodes. We therefore examined the effects of prenatal nicotine exposure on the development of breathing pattern and the ventilatory response to hypoxia (7.4% O2) in vivo using whole-body plethysmography at postnatal days 0, P3, P9, P19 and adult. To determine whether differences observed in vivo were due to altered activity of medullary respiratory networks, motoneurons (MNs) controlling the airway, or MN responsive-ness to nicotinic modulation, we recorded in vitro hypoglossal (XII) nerve and MN activity in rhythmic medullary slice preparations from control and nicotine-exposed P3 mice. Foetal mice were exposed to nicotine using osmotic micropumps implanted in the dam at gestational day 10. Developmental changes in respiratory behaviour in vivo were delayed in the youngest nicotine-exposed animals. The high level of apnoea present during normoxia in P0 control animals (frequency of apnoea, fA, 6.7 ± 0.7 min-1, percent of time apnoeic, TA, 29 ± 6%) and nicotine-exposed groups (fA 8.1 ± 1.7 min-1,TA 25 ± 5%) persisted until P3 in the nicotine group but fell significantly in control animals (fA-2.2 ± 0.7 min-1, TA 5 ± 2%). At the onset of hypoxia, fA and TA fell rapidly and remained low throughout hypoxia except in P0 nicotine-exposed animals where they declined initially (fA 1.8 ± 0.5, TA 4 ± 2%) but then rose progressively during the 12 min hypoxic period to final values of 7.1 ± 2.9 min-1and 17 ± 6% respectively. During recovery, apnoea increased in both groups at P0 (fA 10.8 ± 0.8 and 11.3 ± 1.3 min -1; TA 50 ± 6 and 49 ± 5%). By P3, the absolute magnitude of this posthypoxic increase was reduced in control (fA 5.3 ± 0.1 min-1 ; TA 30 ± 7%) but not nicotine-exposed animals (fA 8.5 ± 1.0 min-1 ; T 44 ± 8%). The frequency and variability of the inspiratory-related output in medullary slice preparations from control and nicotine-exposed animals were indistinguishable. The pattern of the inspiratory-related XII nerve burst and inspiratory synaptic currents recorded from XII MNs were also similar. Local application of nicotine (10–100 μM) over the XII nucleus produced a small, TTX-resistant inward current in MNs that reversed near 0 mV, and potentiated XII nerve inspiratory burst amplitude (25 ± 5%). Both actions were hexamethonium-sensitive, suggesting nicotinic receptor involvement in upper airway control. This potentiation was significantly lower in nicotine-exposed animals (14 ± 3%). Results indicate that prenatal nicotine exposure in mice delays development of breathing pattern, increasing incidence of apnoea in P0–P3 animals before, during and after hypoxia. This increased apnoea does not reflect alterations in the baseline behaviour of rhythm generating or pattern forming circuits, but may result from reduced nicotinic modulation of XII MN activity.

Distribution of corticospinal motor neurons in the postnatal rat: quantitative evidence for massive collateral elimination and modest cell death.

The postnatal development of rat corticospinal motor neurons (CSMN) was studied by retrograde tracing with cholera toxin B subunit (CTB) injected into the upper cervical dorsal spinal cord on the first postnatal day (P0), P3, P10, P20, and at adulthood. CTB-labeled neurons were visualized by immunocytochemistry and extensively quantified throughout the cortex. At P0, CSMN were found to an extent similar to that reported in P3 animals with other neuronal tracers, now permitting in vitro studies of neonatal CSMN. Between P0 and P3, the number of labeled neurons increased by 30% to a total maximum of approximately 185,000 in both cortices. The increase occurred throughout the cortex. At P10, the number of labeled CSMN had decreased to 60% of the number at P3. Fewer CSMN were evident particularly in the perirhinal cortex. Between P10 and P20, the number of CSMN decreased further to 52% of the maximal number at P3. This decrease occurred predominantly in the cingulate and parietal cortex. The number of labeled CSMN in rats injected at P0 and analyzed at P20 was 10% lower than the number in P0-injected littermates that were analyzed at P3, which suggests that only a small portion of the "disappearing" CSMN undergoes developmental neuronal death. Thus, the spinal projection of the remaining 38% is apparently eliminated between P3 and P20. Detailed quantitative analysis of the CSMN distribution demonstrated that neuronal death occurs predominantly in the perirhinal cortex. In contrast, axonal elimination of corticospinal projections occurred throughout the CSMN field, i.e., primarily in the frontal, occipital, and perirhinal cortex between P3-P10 and in the cingulate and parietal cortex between P10-P20.