RESUMEN

Preterm birth results in an increased risk of neonatal brain injury and neurobehavioural disorders. Despite the seriousness of these adverse outcomes, there are currently no effective therapies to protect the vulnerable developing brain. We propose that neurosteroid replacement therapy may be a novel approach in reducing detrimental neurological outcomes following preterm birth. The use of guinea pig primary neuronal and oligodendrocyte cultures with relevance to late gestation allows insight into the mechanisms behind the effectiveness of these treatments. Primary neuronal and oligodendrocyte cultures were derived from fetal guinea pig frontal cortex brain tissue at gestational age 62 (GA62). Cell cultures were pre-treated with either etifoxine (5 µM) or zuranolone (1 µm) for 24 h prior to insult. Cells were then exposed to either oxygen-glucose deprivation (OGD; 0% O2 and no glucose DMEM; preterm birth insult) or sham (standard cell culture conditions; 25 mM DMEM) for 2 h. Lactate dehydrogenase assay (LDH) was performed following OGD as a measure of cytotoxicity. Relative mRNA expression of key neuronal and oligodendrocyte markers, as well as neuronal receptors and transporters, were quantified using high throughput (Fluidigm) RT-PCR. OGD significantly increased cellular cytotoxicity in both neurons and oligodendrocytes. Additionally, key neuronal marker mRNA expression was reduced following OGD, and oligodendrocytes displayed arrested mRNA expression of key markers of lineage progression. Treatment with etifoxine restored a number of parameters back to control levels, whereas treatment with zuranolone provided a robust improvement in all parameters examined. This study has demonstrated the neuroprotective potential of neurosteroid replacement therapy in a model of hypoxia related to preterm birth. Neuroprotection appears to be mediated through glutamate reduction and increased brain derived neurotrophic factor (BDNF). Future work is warranted in examining these treatments in vivo, with the overall aim to suppress preterm associated brain damage and reduce long term outcomes for affected offspring.

RESUMEN

Primary cell culture is a technique that is widely used in neuroscience research to investigate mechanisms that underlie pathologies at a cellular level. Typically, mouse or rat tissue is used for this process; however, altricial rodent species have markedly different neurodevelopmental trajectories comparatively to humans. The use of guinea pig brain tissue presents a novel aspect to this routinely used cell culture method whilst also allowing for dual isolation of two major cell types from a physiologically relevant animal model for studying perinatal neurodevelopment. Primary neuronal and oligodendrocyte cell cultures were derived from fetal guinea pig's frontal cortex brain tissue collected at a gestational age of 62 days (GA62), which is a key time in the neuronal and oligodendrocyte development. The major advantage of this protocol is the ability to acquire both neuronal and oligodendrocyte cellular cultures from the frontal cortex of one fetal brain. Briefly, neuronal cells were grown in 12-well plates initially in a 24-h serum-rich medium to enhance neuronal survival before switching to a serum-free media formulation. Oligodendrocytes were first grown in cell culture flasks using a serum-rich medium that enabled the growth of oligodendrocyte progenitor cells (OPCs) on an astrocyte bed. Following confluency, the shake method of differential adhesion and separation was utilized via horizontally shaking the OPCs off the astrocyte bed overnight. Therefore, OPCs were plated in 12-well plates and were initially expanded in media supplemented with growth hormones, before switching to maturation media to progress the lineage to a mature phenotype. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on both cell culture types to analyze key population markers, and the results were further validated using immunocytochemistry. Primary neurons displayed the mRNA expression of multiple neuronal markers, including those specific to GABAergic populations. These cells also positively stained for microtubule-associated protein 2 (MAP2; a dendritic marker specific to neurons) and NeuN (a marker of neuronal cell bodies). Primary oligodendrocytes expressed all investigated markers of the oligodendrocyte lineage, with a majority of the cells displaying an immature oligodendrocyte phenotype. This finding was further confirmed with positive oligodendrocyte transcription factor (OLIG2) staining, which serves as a marker for the overall oligodendrocyte population. This study demonstrates a novel method for isolating both neurons and oligodendrocytes from the guinea pig brain tissue. These isolated cells display key markers and gene expression that will allow for functional experiments to occur and may be particularly useful in studying neurodevelopmental conditions with perinatal origins.

RESUMEN

The vulnerability of the fetal and newborn brain to events in utero or at birth that cause damage arising from perturbations of cerebral blood flow and metabolism, such as the accumulation of free radicals and excitatory transmitters to neurotoxic levels, has received considerable attention over the last few decades. Attention has usually been on the damage to cerebral structures, particularly, periventricular white matter. The rapid growth of the cerebellum in the latter half of fetal life in species with long gestations, such as the human and sheep, suggests that this may be a particularly important time for the development of cerebellar structure and function. In this short review, we summarize data from recent studies with fetal sheep showing that the developing cerebellum is particularly sensitive to infectious processes, chronic hypoxia and asphyxia. The data demonstrates that the cerebellum should be further studied in insults of this nature as it responds differently to the remainder of the brain. Damage to this region of the brain has implications not only for the development of motor control and posture, but also for higher cognitive processes and the subsequent development of complex behaviours, such as learning, memory and attention.

Asunto(s)

RESUMEN

Clinically, treatment options where fetal distress is anticipated or identified are limited. Allopregnanolone is an endogenous steroid, that positively modulates the GABA(A) receptor, and that has anti-apoptotic and anti-excitotoxic actions, reducing brain damage in adult animal models of brain injury. We sought to determine if prophylactic treatment of the pregnant female with a single dose of this steroid could reduce birth asphyxia-induced losses in hippocampal function at 5 days of age (P5) in spiny mouse neonates (Acomys cahirinus). At 37 days gestation (term=39 days) and 1h before inducing birth asphyxia, spiny mice dams were injected subcutaneously (0.2 ml) with either 3mg/kg allopregnanolone or 20% w/v ß-cyclodextrin vehicle. One hour later, fetuses were either delivered immediately by caesarean section (control group) or exposed to 7.5 min of in utero asphyxia, causing acidosis and hypoxia. At P5, ex vivo hippocampal plasticity was assessed, or brains collected to determine cell proliferation (proliferating cell nuclear antigen; PCNA) or calcium channel expression (inositol trisphosphate receptor type 1; IP(3)R1) using immunohistochemistry. Allopregnanolone partially prevented the decrease in long term potentiation at P5, and the asphyxia-induced increase in IP(3)R1 expression in CA1 pyramidal neurons. There was no effect of allopregnanolone on the asphyxia induced impairment of the input/output (I/O) curve and paired-pulse facilitation (PPF). In control birth pups, maternal allopregnanolone treatment caused significant changes in short term post-synaptic plasticity and also reduced hippocampal proliferation at P5. These findings show that allopregnanolone can modulate hippocampal development and synaptic function in a normoxic or hypoxic environment, possibly by modifying calcium metabolism. Best practice for treatment dose and timing of treatment will need to be carefully considered.

Asunto(s)

RESUMEN

Celecoxib (Celebrex), an inhibitor of cyclooxygenase-2 (COX-2; prostaglandin-endoperoxide synthase 2; EC 1.14.99.1), is widely used in the treatment of chronic inflammation and pain. COX-2 is constitutively expressed in the testis, where it is responsible for prostaglandin production, so inhibition of this enzyme should have effects on testicular function. The effects of administering celecoxib (oral with feed, 0.15% w/w) for 5 weeks on normal testis function and the response to low dose (0.1 mg/kg body weight) or high dose (5.0 mg/kg) lipopolysaccharide (LPS) were examined in adult male rats. Celecoxib caused a 60% reduction in testicular interstitial fluid (IF) prostaglandin E(2) (PGE(2)) concentrations, accompanied by a compensatory increase in COX-2 mRNA expression. Celecoxib increased IF volume by 30%, but had no effect on testis weight, testis morphology or serum testosterone levels. In the celecoxib-fed rats, the dose-dependent inhibitory effects of LPS on testis weight, IF volume and serum testosterone levels were significantly diminished. However, celecoxib had no effect on COX-2 protein levels or LPS-induced expression of the inflammatory mediators interleukin-1beta, tumour necrosis factor-alpha or inducible nitric-oxide synthase. A similar lack of inhibition of LPS-induced cytokine expression by another COX-2 inhibitor, NS-398, was observed in vitro. These data indicate that celecoxib reduces intratesticular activity of COX-2 (as indicated by PGE(2) levels) and inhibits IF formation in the testis, but has no appreciable effect on steroidogenesis or spermatogenesis, at least in the short term. Celecoxib does not appear to alter the ability of the testis to mount an inflammatory response but opposes the deleterious effects of inflammation on IF formation and testosterone production. These results indicate significant roles for products of the COX-2 pathway in testicular vascular control and steroidogenesis, which may have implications for men with marginal fertility taking celecoxib for extended periods, but also highlight the potential of this drug to ameliorate testicular damage caused by systemic or local inflammation.

Asunto(s)

RESUMEN

Prostaglandins (PGs), particularly PGE(2), have been implicated in the control of testicular steroidogenesis, spermatogenesis, and local immunity. However, virtually nothing is known about the expression or activity of the prostaglandin-endoperoxide synthases (PTGSs; also referred to as the cyclooxygenases), the specific rate-limiting enzymes responsible for PG production, in the adult testis. This activity was investigated in rats under normal conditions and during lipopolysaccharide-induced inflammation using quantitative real-time PCR, in situ hybridization, Western blotting, and PGE(2) measurements by ELISA. The mRNA for both the "constitutive" Ptgs1 and the "inducible" Ptgs2 forms was detected in multiple testicular cell types. Testicular Ptgs2 expression was substantially higher than that of Ptgs1, and testicular production of PGE(2) in vitro was found to be suppressed by a specific PTGS2 inhibitor (NS-398), but not by an inhibitor of PTGS1. Further investigation indicated that 1) PGE(2) production in the adult testis is attributable to constitutive expression of PTGS2 by somatic (Leydig cells and Sertoli cells) and spermatogenic cells; 2) testicular macrophages constitutively produce relatively low levels of PTGS2 and PGE(2) but are the only cell type to respond significantly to an inflammatory stimulus by increasing production of PGE(2); and 3) testicular PTGS2 expression and intratesticular PGE(2) levels are only marginally affected by acute inflammation. These data point toward a previously unanticipated maintenance role for the "inducible" PTGS2 enzyme in normal testicular function, as well as an anomalous response of testicular PTGS2 to inflammatory stimuli. Both observations are consistent with the reduced capacity of the testis to initiate and support inflammatory reactions.

Asunto(s)