Accuracy of Estimated Fetal Weight Assessment in Fetuses with Congenital Diaphragmatic Hernia-Is the Hadlock Formula a Reliable Tool?

Objectives: Congenital diaphragmatic hernia (CDH) is defined as organ protrusion from the abdominal to the thoracic cavity. The Hadlock formula is the most commonly used tool for calculating estimated fetal weight (EFW). The anatomical nature of CDH usually leads to underestimation of the abdominal circumference, resulting in underestimation of fetal weight. Accurate weight estimation is essential before birth for counselling, preparation before surgery and ECMO. The research is made to compare the accuracy of Hadlock's formula and Faschingbauer's formula for fetal weight estimation in CDH fetuses population. Methods: In our study, we investigated differences between EFW and actual birthweight in 42 fetuses with CDH as compared to 80 healthy matched controls. EFW was calculated using the Hadlock formula and a recently introduced formula described by Faschingbauer et al., which was tailored for fetuses with CDH. Additionally, both of the formulas were adjusted for the interval between the ultrasound and delivery for both of the groups. Results: The majority of hernias were left-sided (92.8% vs. 7.2%). EFW adjusted for the interval between the ultrasound and delivery had the highest correlation with the actual birthweight in both, study group and controls. We compared the results for both tools and found the Hadlock formula to predict birthweight in CDH children with a 7.8 ± 5.5% error as compared to 7.9 ± 6.5% error for the Faschingbauer's formula. Conclusions: The Hadlock formula adjusted for the interval between the ultrasound and delivery is a more precise method of calculating EFW in fetuses with CDH. Routine biometry scan using Hadlock's formula remains reliable for predicting birthweight.

Prefrontal cortex pyramidal neurons express functional Nav1.8 tetrodotoxin-resistant sodium currents.

It has been repeatedly proved that Nav1.8 tetrodotoxin (TTX)-resistant sodium currents are expressed in peripheral sensory neurons where they play important role in nociception. There are very few publications that show the presence of TTX-resistant sodium currents in central neurons. The aim of this study was to assess if functional Nav1.8 TTX-resistant sodium currents are expressed in prefrontal cortex pyramidal neurons. All recordings were performed in the presence of TTX in the extracellular solution to block TTX-sensitive sodium currents. The TTX-resistant sodium current recorded in this study was mainly carried by the Nav1.8 sodium channel isoform because the Nav1.9 current was inhibited by the -65 mV holding potential that we used throughout the study. Moreover, the sodium current that we recorded was inhibited by treatment with the selective Nav1.8 inhibitor A-803467. Confocal microscopy experiments confirmed the presence of the Nav1.8 α subunit in prefrontal cortex pyramidal neurons. Activation and steady state inactivation properties of TTX-resistant sodium currents were also assessed in this study and they were similar to activation and inactivation properties of TTX-resistant sodium currents expressed in dorsal root ganglia (DRG) neurons. Moreover, this study showed that carbamazepine (60 µM) inhibited the maximal amplitude of the TTX-resistant sodium current. Furthermore, we found that carbamazepine shifts steady state inactivation curve of TTX-resistant sodium currents toward hyperpolarization. This study suggests that the Nav1.8 TTX-resistant sodium channel is expressed not only in DRG neurons, but also in cortical neurons and may be molecular target for antiepileptic drugs such as carbamazepine.

Activity of TREK-2-like Channels in the Pyramidal Neurons of Rat Medial Prefrontal Cortex Depends on Cytoplasmic Calcium.

TREK-2-like channels in the pyramidal neurons of rat prefrontal cortex are characterized by a wide range of spontaneous activity-from very low to very high-independent of the membrane potential and the stimuli that are known to activate TREK-2 channels, such as temperature or membrane stretching. The aim of this study was to discover what factors are involved in high levels of TREK-2-like channel activity in these cells. Our research focused on the PI(4,5)P2-dependent mechanism of channel activity. Single-channel patch clamp recordings were performed on freshly dissociated pyramidal neurons of rat prefrontal cortexes in both the cell-attached and inside-out configurations. To evaluate the role of endogenous stimulants, the activity of the channels was recorded in the presence of a PI(4,5)P2 analogue (PI(4,5)P2DiC8) and Ca2+. Our research revealed that calcium ions are an important factor affecting TREK-2-like channel activity and kinetics. The observation that calcium participates in the activation of TREK-2-like channels is a new finding. We showed that PI(4,5)P2-dependent TREK-2 activity occurs when the conditions for PI(4,5)P2/Ca2+ nanocluster formation are met. We present a possible model explaining the mechanism of calcium action.

The Effect of L-Ascorbic Acid and Serum Reduction on Tenogenic Differentiation of Human Mesenchymal Stromal Cells.

BACKGROUND AND OBJECTIVES: Despite significant improvement in the treatment of tendon injuries, the full tissue recovery is often not possible because of its limited ability to auto-repair. The transplantation of mesenchymal stromal cells (MSCs) is considered as a novel approach in the treatment of tendinopathies. The question about the optimal culture conditions remains open. In this study we aimed to investigate if serum reduction, L-ascorbic acid supplementation or a combination of both factors can induce tenogenic differentiation of human adipose-derived MSCs (ASCs). METHODS AND RESULTS: Human ASCs from 3 healthy donors were used in the study. The tested conditions were: 0.5 mM of ascorbic acid 2-phosphate (AA-2P), reduced serum content (2% FBS) or combination of these two factors. The combination of AA-2P and 2% FBS was the only experimental condition that caused a significant increase of the expression of all analyzed genes related to tenogenesis (SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3, DECORIN) in comparison to the untreated control (evaluated by RT-PCR, 5th day of experiment). Moreover, this treatment significantly increased the synthesis of SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3 proteins at the same time point (evaluated by Western blot method). Double immunocytochemical staining revealed that AA-2P significantly increased the extracellular deposition of both types of collagens. Semi-quantitative Electron Spin Resonance analysis of ascorbyl free radical revealed that AA-2P do not induce harmful transition metals-driven redox reactions in cell culture media. CONCLUSIONS: Obtained results justify the use of reduced content of serum with the addition of 0.5 mM of AA-2P in tenogenic inducing media.

Mosaic Recombinant Adeno-associated Virus Vector rAAV/DJ/CAG for Targeted Gene Delivery to Melanoma Cells Metastasized to the Lung.

BACKGROUND/AIM: Patients with metastasized melanoma have limited treatment options and poor diagnosis. Therefore, the development of treatments requires a new therapeutic approach, of which gene therapy using rAAV vectors can be proposed. The aim of the study was to examine the efficiency of the rAAV vector to transduce mouse melanoma cells both in vitro and in vivo. MATERIALS AND METHODS: Different rAAV serotypes encoding GFP under the control of both chicken beta-actin and cytomegalovirus promoters were used in the experiments. Intranasal, intraperitoneal, intravenous and intratumoral pathways of administration of rAAV vectors were tested using quantitative-PCR and immunohistochemical staining. RESULTS: The highest transduction efficiency in metastatic cells in vivo was observed 7 days after intranasal administration of a 1010 gc/0.03 ml dose of rAAV/DJ-CAG. CONCLUSION: Melanoma gene therapy based on rAAV vectors is a possible treatment option.

Effects of ß3-adrenergic receptor stimulation on the resting holding current of medial prefrontal cortex pyramidal neurons in young rats.

While the expression of ß3-adrenergic receptors is firmly established in adipose, kidney and heart tissue, their expression and function in the brain remains unclear despite their potential role in depression and stress-related disorders. This study aimed to investigate the expression of ß3-adrenoreceptors and their involvement in the mechanism controlling the resting holding current in layer V medial prefrontal cortex (mPFC) pyramidal neurons in young rats. Applications of the selective ß3-adrenergic receptor agonists BRL 37344 and SR 58611 A evoked inward currents in the tested neurons. The inward currents evoked by BRL 37344 or noradrenaline (a nonselective physiological adrenergic receptor agonist) were prevented or decreased, respectively, by the selective ß3-receptor antagonist L-748,337. Western blot and fluorescence immunohistochemistry analyses revealed ß3-adrenergic receptor protein expression in the mPFC. Thus, based on the results obtained here, functional ß3-adrenergic receptors are expressed in layer V mPFC pyramidal neurons and their activation evokes inward currents.

Distribution of polyethylenimine in zebrafish embryos

Background: Polyethylenimine (PEI) plays important roles in the pharmaceutical design of non-viral gene delivery systems. Due to a set of unique physicochemical properties this cationic polymer has a great potential in modern gene therapies. Objective: The aim of the present study was to determine the distribution of branched PEI (0.8 kDa) in zebrafish embryos (Danio rerio). Material and methods: Zebrafish embryos at 3 hours post-fertilization (hpf) were incubated with PEI (10 µg/ml) for 24 and 48 hours and studied using the confocal laser microscopy. Results: The obtained results show that PEI effectively distributed into the layers of the chorion and yolk sac in developing embryos due to first 24 hours of exposure. In contrast, PEI was found in the yolk, head, trunk and tail of the embryos due to prolonged treatments (48 hours). Conclusion: The study evidences a high distribution of the branched PEI (0.8 kDa) polymer in the zebrafish embryo tissues.

New Insight into the Synthesis and Biological Activity of the Polymeric Materials Consisting of Folic Acid and ß-Cyclodextrin.

This work presents a very new look at folate targeting and is focused on synthesizing and assessing the biological activity of folic acid-targeted drug delivery materials based on ß-cyclodextrin. Both folic acid and ß-cyclodextrin have been covalently conjugated to branched polyethylenimine as the polymeric vector. Host-guest inclusion of folic acid into a ß-cyclodextrin cavity, demonstrated by means of the spectroscopic methods (2-D NMR, IR, UV-Vis), is found to be of crucial importance for biological activity of nanotherapeutics. This paper describes the very first example of the versatile synthetic approach to create the polymeric biosystems, where folic acid activity is not limited by the inclusion phenomenon. Cytotoxicity of the obtained polymeric materials against Lewis lung carcinoma cells is determined by neutral red uptake assay. Folate receptor-binding studies reveal that the developed synthetic approach enables full exploitation of the potential of folic acid as a targeting ligand.

Noradrenaline Modulates the Membrane Potential and Holding Current of Medial Prefrontal Cortex Pyramidal Neurons via ß1-Adrenergic Receptors and HCN Channels.

The medial prefrontal cortex (mPFC) receives dense noradrenergic projections from the locus coeruleus. Adrenergic innervation of mPFC pyramidal neurons plays an essential role in both physiology (control of memory formation, attention, working memory, and cognitive behavior) and pathophysiology (attention deficit hyperactivity disorder, posttraumatic stress disorder, cognitive deterioration after traumatic brain injury, behavioral changes related to addiction, Alzheimer's disease and depression). The aim of this study was to elucidate the mechanism responsible for adrenergic receptor-mediated control of the resting membrane potential in layer V mPFC pyramidal neurons. The membrane potential or holding current of synaptically isolated layer V mPFC pyramidal neurons was recorded in perforated-patch and classical whole-cell configurations in slices from young rats. Application of noradrenaline (NA), a neurotransmitter with affinity for all types of adrenergic receptors, evoked depolarization or inward current in the tested neurons irrespective of whether the recordings were performed in the perforated-patch or classical whole-cell configuration. The effect of noradrenaline depended on ß1- and not α1- or α2-adrenergic receptor stimulation. Activation of ß1-adrenergic receptors led to an increase in inward Na+ current through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which carry a mixed Na+/K+ current. The protein kinase A- and C-, glycogen synthase kinase-3ß- and tyrosine kinase-linked signaling pathways were not involved in the signal transduction between ß1-adrenergic receptors and HCN channels. The transduction system operated in a membrane-delimited fashion and involved the ßγ subunit of G-protein. Thus, noradrenaline controls the resting membrane potential and holding current in mPFC pyramidal neurons through ß1-adrenergic receptors, which in turn activate HCN channels via a signaling pathway involving the ßγ subunit.

Hypertension in rats is associated with an increased permeability of the colon to TMA, a gut bacteria metabolite.

An increased blood trimethylamine N-oxide (TMAO) has emerged as a marker of cardiovascular mortality, however, the mechanisms of the increase are not clear. We evaluated if hypertension was associated with changes in the colon permeability to trimethylamine (TMA), a TMAO precursor. We did experiments on male, 24-26-week-old normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and SHR treated with enalapril, an antihypertensive drug (SHR-E). To check the colon permeability and liver TMA clearance, blood was collected from the portal vein and hepatic veins confluence, at baseline and after the intracolonic administration of TMA. Arterial blood pressure (BP) and intestinal blood flow (IBF) recordings and histological assessment of the colon were performed. SHR showed an increased gut-blood barrier permeability to TMA. Namely, at baseline SHR had a higher BP and portal blood TMA, but a lower IBF than WKY. After the intracolonic administration of TMA, SHR had a significantly higher portal blood TMA and higher TMA liver clearance than WKY. In SHR the arteriolar walls of the colon mucosa were significantly thicker than in WKY. Furthermore, SHR showed a significant decrease in the height of the mucosa. In contrast, SHR-E had lower portal blood TMA, lower BP and smaller thickness of arteriolar walls, but higher IBF than SHR, which indicates improved function of the gut-blood barrier in SHR-E. All groups had similar immunostaining of occludin and zonula occludens-1, markers of tight junctions. In conclusion, hypertensive rats show an increased permeability of the colon to TMA, which is accompanied by morphological and hemodynamic alterations in the colon. Therefore, cardiovascular diseases may be characterized by an increased permeability of the gut-blood barrier to bacterial metabolites such as TMA.

Age-dependent expression of Nav1.9 channels in medial prefrontal cortex pyramidal neurons in rats.

Developmental changes that occur in the prefrontal cortex during adolescence alter behavior. These behavioral alterations likely stem from changes in prefrontal cortex neuronal activity, which may depend on the properties and expression of ion channels. Nav1.9 sodium channels conduct a Na+ current that is TTX resistant with a low threshold and noninactivating over time. The purpose of this study was to assess the presence of Nav1.9 channels in medial prefrontal cortex (mPFC) layer II and V pyramidal neurons in young (20-day old), late adolescent (60-day old), and adult (6- to 7-month old) rats. First, we demonstrated that layer II and V mPFC pyramidal neurons in slices obtained from young rats exhibited a TTX-resistant, low-threshold, noninactivating, and voltage-dependent Na+ current. The mRNA expression of the SCN11a gene (which encodes the Nav1.9 channel) in mPFC tissue was significantly higher in young rats than in late adolescent and adult rats. Nav1.9 protein was immunofluorescently labeled in mPFC cells in slices and analyzed via confocal microscopy. Nav1.9 immunolabeling was present in layer II and V mPFC pyramidal neurons and was more prominent in the neurons of young rats than in the neurons of late adolescent and adult rats. We conclude that Nav1.9 channels are expressed in layer II and V mPFC pyramidal neurons and that Nav1.9 protein expression in the mPFC pyramidal neurons of late adolescent and adult rats is lower than that in the neurons of young rats. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1371-1384, 2017.

Renal denervation decreases blood pressure and renal tyrosine hydroxylase but does not augment the effect of hypotensive drugs.

The effect of renal denervation on the efficacy of antihypertensive drugs has not yet been elucidated. Twenty-week-old spontaneously hypertensive rats were treated with metoprolol, losartan, indapamide, or saline (controls) and assigned to renal denervation or a sham procedure. Acute hemodynamic measurements were performed ten days later. Series showing a significant interaction between renal denervation and the drugs were repeated with chronic telemetry measurements. In the saline series, denervated rats showed a significantly lower mean arterial blood pressure (blood pressure) than the sham-operated rats. In contrast, in the metoprolol series denervated rats showed a significantly higher blood pressure than sham rats. There were no differences in blood pressure between denervated and sham rats in the losartan and indapamide series. In chronic studies, a 4-week treatment with metoprolol caused a decrease in blood pressure. Renal denervation and sham denervation performed 10 days after the onset of metoprolol treatment did not affect blood pressure. Denervated rats showed markedly reduced renal nerve tyrosine hydroxylase levels. In conclusion, renal denervation decreases blood pressure in hypertensive rats. The hypotensive action of metoprolol, indapamide, and losartan is not augmented by renal denervation, suggesting the absence of synergy between renal denervation and the drugs investigated in this study.

The extracellular matrix glycoprotein tenascin-C and matrix metalloproteinases modify cerebellar structural plasticity by exposure to an enriched environment.

The importance of the extracellular matrix (ECM) glycoprotein tenascin-C (TnC) and the ECM degrading enzymes, matrix metalloproteinases (MMPs) -2 and -9, in cerebellar histogenesis is well established. This study aimed to examine whether there is a functional relationship between these molecules in regulating structural plasticity of the lateral deep cerebellar nucleus. To this end, starting from postnatal day 21, TnC- or MMP-9-deficient mice were exposed to an enriched environment (EE). We show that 8 weeks of exposure to EE leads to reduced lectin-based staining of perineuronal nets (PNNs), reduction in the size of GABAergic and increase in the number and size of glutamatergic synaptic terminals in wild-type mice. Conversely, TnC-deficient mice showed reduced staining of PNNs compared to wild-type mice maintained under standard conditions, and exposure to EE did not further reduce, but even slightly increased PNN staining. EE did not affect the densities of the two types of synaptic terminals in TnC-deficient mice, while the size of inhibitory, but not excitatory synaptic terminals was increased. In the time frame of 4-8 weeks, MMP-9, but not MMP-2, was observed to influence PNN remodeling and cerebellar synaptic plasticity as revealed by measurement of MMP-9 activity and colocalization with PNNs and synaptic markers. These findings were supported by observations on MMP-9-deficient mice. The present study suggests that TnC contributes to the regulation of structural plasticity in the cerebellum and that interactions between TnC and MMP-9 are likely to be important for these processes to occur.

[Standardization of the quantitative flow cytometric test with anti-D antibodies for fetomaternal hemorrhage in RhD negative women]. / Standaryzacja metody ilosciowej oceny przecieku plodowo-matczynego u kobiet RhD ujemnych przy pomocy cytometrii przeplywowej z uzyciem przeciwcial anty-D.

BACKGROUND: In order to determine the appropriate dose of anti-D immunoglobulin to be administered as a preventive measure against hemolytic disease of the fetus/newborn in the subsequent pregnancy it is necessary to assess the number of fetal red blood cells that infiltrate/penetrate into the maternal circulation as a result of fetomaternal hemorrhage (FMH). One of the quantitative methods of FMH analysis is based on flow cytometry (FACS) which makes use of monoclonal antibodies to RhD antigen (anti-D test). The aim of the study was to further develop the method, evaluate its sensitivity and reproducibility and to compare it with the test based on the detection of fetal hemoglobin (HbF). MATERIAL AND METHODS: The FACS study involved 20 RhD negative pregnant women and 80 RhD negative women after delivery. The following monoclonal antibodies were used: BRAD 3 FITC (anti-RhD antigen), CD45 PerCP (anti leukocyte antigen CD45), and anti-HbF PE. RESULTS: The fluorescence intensity of cells incubated with BRAD 3 FITC was demonstrated to depend on the RhD antigen expression, though the anti-D test also detects the weak D variant. The CD45 PerCP antibodies increased the sensitivity of anti-D test since they eliminated the leukocytes which non-specifically bind anti-D from the analysis. The presence of anti-D antibodies in maternal plasma does not affect the quantitative assessment of the fetal RhD positive fetal cells with BRAD 3 FITC. In case of FMH, the results of the anti-D test were similar to those with anti-HbF antibodies. CONCLUSIONS: The flow cytometric test with anti-D and anti-CD45 is useful in the assessment of the fetomaternal hemorrhage in RhD negative women. The sensitivity of the test is estimated at 0.05%.

Epidemiological and clinical reasons for vaccination against pertussis and influenza in pregnant women.

Vaccinations in pregnancy are an important aspect of prenatal care for improving both maternal health and neonatal outcomes. Despite the fact that protection against some infectious diseases for pregnant women can be easily provided through immunizations, current coverage rates are low. Two vaccines are notably recommended during pregnancy: influenza and the combined tetanus, diphtheria and acellular pertussis (Tdap) vaccine. In this review the authors discuss current recommendations for vaccination against pertussis and influenza in pregnant women in terms of epidemiological, clinical, and immunological reasons, taking into account safety and effectiveness. Promoting patients' awareness about pertussis and influenza and encouraging general practitioners, nurses and obstetricians to recommend the pertussis booster and influenza vaccine will hopefully increase the number of pregnant women who choose to become vaccinated.

Reward learning requires activity of matrix metalloproteinase-9 in the central amygdala.

Learning how to avoid danger and pursue reward depends on negative emotions motivating aversive learning and positive emotions motivating appetitive learning. The amygdala is a key component of the brain emotional system; however, an understanding of how various emotions are differentially processed in the amygdala has yet to be achieved. We report that matrix metalloproteinase-9 (MMP-9, extracellularly operating enzyme) in the central nucleus of the amygdala (CeA) is crucial for appetitive, but not for aversive, learning in mice. The knock-out of MMP-9 impairs appetitively motivated conditioning, but not an aversive one. MMP-9 is present at the excitatory synapses in the CeA with its activity greatly enhanced after the appetitive training. Finally, blocking extracellular MMP-9 activity with its inhibitor TIMP-1 provides evidence that local MMP-9 activity in the CeA is crucial for the appetitive, but not for aversive, learning.

Matrix metalloproteinase 9 regulates cell death following pilocarpine-induced seizures in the developing brain.

Matrix metalloproteinases (MMPs) are involved in tissue repair, cell death and morphogenesis. We investigated the role of the gelatinases MMP-2 and MMP-9 in the pathogenesis of neuronal death induced by prolonged seizures in the developing brain. Seven-day-old rats, MMP-9 knockout mice and transgenic rats overexpressing MMP-9 received intraperitoneal injections of pilocarpine, 250 mg/kg, to induce seizures. After 6-72 h pups were sacrificed, tissue from different brain regions was isolated and expression of MMP-9 mRNA and protein was analyzed by real-time PCR or Western blot. Additionally, brains were fixed and processed for TUNEL-staining, immunohistochemistry and in situ zymography. We found increased numbers of TUNEL-positive cells 24 h after pilocarpine-induced seizures, most pronounced in cortical areas and the dentate gyrus, and less pronounced in thalamus. At 6-24 h, MMP-9 mRNA levels showed significant elevation compared to sham-treated controls; this effect resolved by 48 h, whereas MMP-2 mRNA levels remained stable. Cortical gelatinolytic activity, monitored by in situ zymography, was enhanced following pilocarpine-induced seizures. The MMP inhibitor GM 6001 ameliorated cell death following pilocarpine-induced seizures in infant rats. MMP-9 knockout mice were less susceptible to seizure-induced brain injury. Transgenic rats overexpressing MMP-9 were equally susceptible to seizure-induced brain injury as wild type rats. Our results suggest a significant contribution of MMP-9 to cell death after pilocarpine-induced seizures in the developing brain. As indicated by Western blot analysis, MMP-9 activation may be linked to activation of the Erk/CREB-pathway. The findings implicate involvement of MMP-9 in the pathophysiology of brain injury following seizures in the developing brain.

Experience-dependent plasticity and modulation of growth regulatory molecules at central synapses.

Structural remodeling or repair of neural circuits depends on the balance between intrinsic neuronal properties and regulatory cues present in the surrounding microenvironment. These processes are also influenced by experience, but it is still unclear how external stimuli modulate growth-regulatory mechanisms in the central nervous system. We asked whether environmental stimulation promotes neuronal plasticity by modifying the expression of growth-inhibitory molecules, specifically those of the extracellular matrix. We examined the effects of an enriched environment on neuritic remodeling and modulation of perineuronal nets in the deep cerebellar nuclei of adult mice. Perineuronal nets are meshworks of extracellular matrix that enwrap the neuronal perikaryon and restrict plasticity in the adult CNS. We found that exposure to an enriched environment induces significant morphological changes of Purkinje and precerebellar axon terminals in the cerebellar nuclei, accompanied by a conspicuous reduction of perineuronal nets. In the animals reared in an enriched environment, cerebellar nuclear neurons show decreased expression of mRNAs coding for key matrix components (as shown by real time PCR experiments), and enhanced activity of matrix degrading enzymes (matrix metalloproteinases 2 and 9), which was assessed by in situ zymography. Accordingly, we found that in mutant mice lacking a crucial perineuronal net component, cartilage link protein 1, perineuronal nets around cerebellar neurons are disrupted and plasticity of Purkinje cell terminal is enhanced. Moreover, all the effects of environmental stimulation are amplified if the afferent Purkinje axons are endowed with enhanced intrinsic growth capabilities, induced by overexpression of GAP-43. Our observations show that the maintenance and growth-inhibitory function of perineuronal nets are regulated by a dynamic interplay between pre- and postsynaptic neurons. External stimuli act on this interaction and shift the balance between synthesis and removal of matrix components in order to facilitate neuritic growth by locally dampening the activity of inhibitory cues.

Matrix metalloproteinases 2 and 9 fail to influence drug-induced neuroapoptosis in developing rat brain.

Matrix metalloproteinases (MMPs) play an essential role in tissue repair, cell death, and morphogenesis. The aim of the present study was to investigate potential involvement of selected MMPs in the pathogenesis of neuronal apoptosis induced by the NMDA antagonist MK-801 (dizocilpine) or the GABA(A) agonist phenobarbital in infant rats, transgenic rats overexpressing MMP-9 and MMP-9 knockout mice. Seven-day-old rats or knockout mice received intraperitoneal injections of MK-801, 1 mg/kg, or phenobarbital, 50 mg/kg. At different survival intervals following administration of the compounds (1-72 h), pups were sacrificed, tissue from different brain regions was isolated, and the expression and activity of MMP-2 and MMP-9 were analyzed by real-time PCR, western blot, and zymography. In addition, brains were fixed and processed for TUNEL staining. In all the brain regions analyzed, we found an increased number of TUNEL-positive cells 24 h after administration of MK-801. After treatment, we detected no significant increase in MMP-2 or MMP-9 mRNA expression in cortical areas. No changes in the MMP-9 protein expression or gelatinolytic activity of MMP-2 were observed in conjunction with MK-801 or phenobarbital-induced neuroapoptosis in any brain region analyzed. The extent of neurodegeneration induced by MK-801 or phenobarbital was not altered in MMP-9 transgenic rats and was increased in MMP-9 knockout mice compared to wild-type rats and mice. Treatment with the panmetalloproteinase inhibitor GM6001 did not confer protection against MK-801-induced apoptotic cell death in the developing rat brain. Our results suggest that activation of MMP-9 and MMP-2 does not contribute to pathogenesis of neuronal apoptosis caused by NMDA antagonists or GABA(A) agonists in the developing rat and mouse brain.

CD44 is expressed in non-myelinating Schwann cells of the adult rat, and may play a role in neurodegeneration-induced glial plasticity at the neuromuscular junction.

CD44 is a multifunctional cell surface glycoprotein which regulates cell-cell and cell-matrix interactions in a variety of tissues. In particular, the protein was found to be expressed in glial cells of developing, but not adult, peripheral nerves, where it takes part in signaling mediated by ErbB class of receptors for neuregulins. Here, we demonstrate, using high resolution morphological methods, tissue fractionation and RT-PCR, that CD44 is strongly expressed in terminal Schwann cell (TSC) at the neuromuscular junction (NMJ) of the adult rat skeletal muscle. As CD44 is also expressed by Schwann cells of the non-myelinated Remak bundles of the proximal peripheral nerves, it appears to be a marker of non-myelinating Schwann cell subpopulation. The analysis of transgenic rats bearing a mutated superoxide-dismutase gene (SOD1(G93A)) causing familial amyotrophic lateral sclerosis (ALS) revealed that TSC activation and morphological plasticity at the NMJ, caused by ongoing denervation-reinnervation is associated with a strong increase in CD44 expression therein. Notably, CD44 immunoreactivity is present in fine axon-escheating processes of the glial cells that guide reinnervation. In addition, we found that both in normal and SOD1(G93A) muscle, CD44 expressed in TSC partially colocalizes with immunoreactivities of neuregulin receptors ErbB2 and ErbB3. The colocalization appears to reflect a physical interaction, as evidenced by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) analysis between CD44 and ErbB3. Importantly, TSC activation upon ALS-like neurodegeneration results in significant increase in molecular proximity of CD44 and ErbB3, which may have an impact on glial plasticity at the NMJ.