Neurofilament Expression Research Articles

Abstract 4913: Investigation of the in vitro differentiation ability of hybrid cell clones derived from spontaneous cell fusion events between murine breast cancer cells and murine mesenchymal stem cells.

Abstract We have previously shown that murine breast cancer cell lines are capable of acquiring malignant attributes through cell fusion with stem cell lines in vitro. Hybrid cell clones emerged from spontaneous fusion events in a co-culture model showed an increased resistance against chemotherapeutic substances together with a higher proliferation rate and genetic alterations compared to their parental cell lines. Since solid tumors, including breast cancer, have pronounced heterogeneity of cell populations, cell fusion between cancer cells and tumor infiltrating immune cells as well as stem cells is one mechanism how tumor cells could acquire new properties. With relation to the importance of cell fusion in tumor heterogeneity and progression the objective of this study was to characterize and investigate if hybrid cell clones arised from spontaneous fusion events between hygromycin resistant murine 67NR-Hyg mammary carcinoma cells and puromycin resistant murine bone marrow derived stem cells (BMDCs) from Tg(GFPU)5Nagy/J mice to maintain their ability to differentiate into the neural and osteogenic lineage. Dual hygromycin/puromycin resistant mBMDC/67NRHyg cells originated by cell fusion were confirmed by a dual drug selection procedure and PCR analysis to examine an overlap of parental markers. BMDCs are generally defined as self-renewable, multipotent progenitor cells with the ability to differentiate into several mesenchymal lineages. Differentiation was performed by cultivating the cell clones and parental cell lines under appropriate conditions to an established neural and osteogenic differentiation protocol, respectively. Our results revealed through molecular and biochemical analyses that hybrid cell clones and parental cell lines were positive for neuronal as well as osteogenic markers. Differentiation of hybrid cell clones into the osteogenic lineage was associated with expression of osteocalcin, bone sialoprotein II and collagen type I alpha 1 and increasing alkaline phosphatase activity, while differentiation into neuronal lineage was concomitant with expression of neurofilament M, GAD67 and class III beta-tubulin. In conclusion, our data show that hybrid cell clones are able to differentiate into specialized cells from both osteogenic and neuronal lineages. These stem cell properties indicate that cell clones derived from cell fusion events maintain abilities of the parental cell lines. Citation Format: Marieke Mohr, Kurt S. Zaenker, Thomas Dittmar. Investigation of the in vitro differentiation ability of hybrid cell clones derived from spontaneous cell fusion events between murine breast cancer cells and murine mesenchymal stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4913. doi:10.1158/1538-7445.AM2013-4913

Combination of fibrin-agarose hydrogels and adipose-derived mesenchymal stem cells for peripheral nerve regeneration

Objective. The objective was to study the effectiveness of a commercially available collagen conduit filled with fibrin-agarose hydrogels alone or with fibrin-agarose hydrogels containing autologous adipose-derived mesenchymal stem cells (ADMSCs) in a rat sciatic nerve injury model. Approach. A 10 mm gap was created in the sciatic nerve of 48 rats and repaired using saline-filled collagen conduits or collagen conduits filled with fibrin-agarose hydrogels alone (acellular conduits) or with hydrogels containing ADMSCs (ADMSC conduits). Nerve regeneration was assessed in clinical, electrophysiological and histological studies. Main results. Clinical and electrophysiological outcomes were more favorable with ADMSC conduits than with the acellular or saline conduits, evidencing a significant recovery of sensory and motor functions. Histological analysis showed that ADMSC conduits produce more effective nerve regeneration by Schwann cells, with higher remyelination and properly oriented axonal growth that reached the distal areas of the grafted conduits, and with intensely positive expressions of S100, neurofilament and laminin. Extracellular matrix was also more abundant and better organized around regenerated nerve tissues with ADMSC conduits than those with acellular or saline conduits. Significance. Clinical, electrophysiological and histological improvements obtained with tissue-engineered ADMSC conduits may contribute to enhancing axonal regeneration by Schwann cells.

The effect of exercise on mobilization of hematopoietic progenitor cells involved in the repair of sciatic nerve crush injury [RETRACTED

Object Mobilization of hematopoietic progenitor cells (HPCs) from bone marrow involved in the process of peripheral nerve regeneration occurs mostly through deposits of CD34(+) cells. Treadmill exercise, with either differing intensity or duration, has been shown to increase axon regeneration and sprouting, but the effect of mobilization of HPCs on peripheral nerve regeneration due to treadmill exercise has not yet been elucidated. Methods Peripheral nerve injury was induced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were categorized into 2 groups: those with and without treadmill exercise (20 m/min for 60 minutes per day for 7 days). Cytospin and flow cytometry were used to determine bone marrow progenitor cell density and distribution. Neurobehavioral analysis, electrophysiological study, and regeneration marker expression were investigated at 1 and 3 weeks after exercise. The accumulation of HPCs, immune cells, and angiogenesis factors in injured nerves was determined. A separate chimeric mice study was conducted to assess CD34(+) cell distribution according to treadmill exercise group. Results Treadmill exercise significantly promoted nerve regeneration. Increased Schwann cell proliferation, increased neurofilament expression, and decreased Schwann cell apoptosis were observed 7 days after treadmill exercise. Elevated expression of S100 and Luxol fast blue, as well as decreased numbers of vacuoles, were identified in the crushed nerve 3 weeks after treadmill exercise. Significantly increased numbers of mononuclear cells, particularly CD34(+) cells, were induced in bone marrow after treadmill exercise. The deposition of CD34(+) cells was abolished by bone marrow irradiation. In addition, deposits of CD34(+) cells in crushed nerves paralleled the elevated expressions of von Willebrand factor, isolectin B4, and vascular endothelial growth factor. Conclusions Bone marrow HPCs, especially CD34(+) cells, were able to be mobilized by low-intensity treadmill exercise, and this effect paralleled the significant expression of angiogenesis factors. Treadmill exercise stimulation of HPC mobilization during peripheral nerve regeneration could be used as a therapy in human beings.

Brain Activity of Thioctic Acid Enantiomers: In Vitro and in Vivo Studies in an Animal Model of Cerebrovascular Injury

Oxidative stress is an imbalance between the production of free radicals and antioxidant defense mechanisms, potentially leading to tissue damage. Oxidative stress has a key role in the development of cerebrovascular and/or neurodegenerative diseases. This phenomenon is mainly mediated by an enhanced superoxide production by the vascular endothelium with its consequent dysfunction. Thioctic, also known as alpha-lipoic acid (1,2-dithiolane-3-pentanoic acid), is a naturally occurring antioxidant that neutralizes free radicals in the fatty and watery regions of cells. Both the reduced and oxidized forms of the compound possess antioxidant ability. Thioctic acid has two optical isomers designated as (+)- and (−)-thioctic acid. Naturally occurring thioctic acid is the (+)-thioctic acid form, but the synthetic compound largely used in the market for stability reasons is a mixture of (+)- and (−)-thioctic acid. The present study was designed to compare the antioxidant activity of the two enantiomers versus the racemic form of thioctic acid on hydrogen peroxide-induced apoptosis in a rat pheochromocytoma PC12 cell line. Cell viability was evaluated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and free oxygen radical species (ROS) production was assessed by flow cytometry. Antioxidant activity of the two enantiomers and the racemic form of thioctic acid was also evaluated in spontaneously hypertensive rats (SHR) used as an in vivo model of increased oxidative stress. A 3-h exposure of PC12 cells to hydrogen peroxide (H2O2) significantly decreased cell viability and increased levels of intracellular ROS production. Pre-treatment with racemic thioctic acid or (+)-enantiomer significantly inhibited H2O2-induced decrease in cell viability from the concentration of 50 μmol/L and 20 μmol/L, respectively. Racemic thioctic acid and (+)-salt decreased levels of intracellular ROS, which were unaffected by (−)-thioctic acid. In the brain of SHR, the occurrence of astrogliosis and neuronal damage, with a decreased expression of neurofilament 200 kDa were observed. Treatment of SHR for 30 days with (+)-thioctic acid reduced the size of astrocytes and increased the neurofilament immunoreaction. The above findings could contribute to clarify the role played by thioctic acid in central nervous system injury related to oxidative stress. The more pronounced effect of (+)-thioctic acid observed in this study may have practical therapeutic implications worthy of being investigated in further preclinical and clinical studies.

Multinodular and Vacuolating Neuronal Tumors of the Cerebrum: 10 Cases of a Distinctive Seizure‐Associated Lesion

We report 10 cases of a non-neurocytic, purely neuronal tumor affecting adults. Situated in the cerebral hemispheres, with 7 of 10 confined to the temporal lobes, most presented with seizures as their principal clinical manifestations. On magnetic resosnance imaging (MRI), the tumors generally appeared solid and non-contrast enhancing with minimal diffuse infiltration, edema, or mass effect. Six examples demonstrated internal nodularity. Microscopically, the tumor cells were largely distributed into discrete and coalescent nodules exhibiting varying degrees of matrix vacuolization, principally within the deep cortical ribbon and superficial subcortical white matter. Populating elements ranged from morphologically ambiguous to recognizably neuronal, with only two cases manifesting overt ganglion cell cytology. In all cases, tumor cells exhibited widespread nuclear immunolabeling for the HuC/HuD neuronal antigens, although expression of other neuronal markers, including synaptophysin, neurofilament and chromogranin was variable to absent. Tumor cells also failed to express GFAP, p53, IDH1 R132H, or CD34, although CD34-labeling ramified neural elements were present in the adjoining cortex of seven cases. Molecular analysis in a subset of cases failed to reveal DNA copy number abnormalities or BRAF V600E mutation. Follow-up data indicate that this unusual neuronal lesion behaves in benign, World Health Organization (WHO) grade I fashion and is amenable to surgical control.

Влияние воспаления на характер экспрессии нейрофиламента в поджелудочной железе при хроническом панкреатите

Влияние воспаления на характер экспрессии нейрофиламента в поджелудочной железе при хроническом панкреатите

The Effects of Target Skeletal Muscle Cells on Dorsal Root Ganglion Neuronal Outgrowth and Migration In Vitro

Targets of neuronal innervations play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. During development, neurons extend axons to their targets, and then their survival become dependent on the trophic substances secreted by their target cells. Sensory endings were present on myoblasts, myotubes, and myofibers in all intrafusal bundles regardless of age. The interdependence of sensory neurons and skeletal muscle (SKM) cells during both embryonic development and the maintenance of the mature functional state has not been fully understood. In the present study, neuromuscular cocultures of organotypic dorsal root ganglion (DRG) explants and dissociate SKM cells were established. Using this culture system, the morphological relationship between DRG neurons and SKM cells, neurites growth and neuronal migration were investigated. The migrating neurons were determined by fluorescent labeling of microtubule-associated protein-2 (MAP-2) and neurofilament 200 (NF-200) or growth-associated protein 43 (GAP-43). The expression of NF-200 and GAP-43 and their mRNAs was evaluated by Western blot assay and real time-PCR analysis. The results reveal that DRG explants showed more dense neurites outgrowth in neuromuscular cocultures as compared with that in the culture of DRG explants alone. The number of total migrating neurons (the MAP-2-expressing neurons) and the percentage NF-200-immunoreactive (IR) and GAP-43-IR neurons increased significantly in the presence of SKM cells. The levels of NF-200 and GAP-43 and their mRNAs increased significantly in neuromuscular cocultures as compared with that in the culture of DRG explants alone. These results suggested that target SKM cells play an important role in regulating neuronal protein synthesis, promoting neuritis outgrowth and neuronal migration of DRG explants in vitro. These results not only provide new clues for a better understanding of the association of SKM cells with DRG sensory neurons during development, they may also have implications for axonal regeneration after nerve injury.

Song Bu Li Decoction, a Traditional Uyghur Medicine, Protects Cell Death by Regulation of Oxidative Stress and Differentiation in Cultured PC12 Cells

Song Bu Li decoction (SBL) is a traditional Uyghur medicinal herbal preparation, containing Nardostachyos Radix et Rhizoma. Recently, SBL is being used to treat neurological disorders (insomnia and neurasthenia) and heart disorders (arrhythmia and palpitation). Although this herbal extract has been used for many years, there is no scientific basis about its effectiveness. Here, we aimed to evaluate the protective and differentiating activities of SBL in cultured PC12 cells. The pretreatment of SBL protected the cell against tBHP-induced cell death in a dose-dependent manner. In parallel, SBL suppressed intracellular reactive oxygen species (ROS) formation. The transcriptional activity of antioxidant response element (ARE), as well as the key antioxidative stress proteins, was induced in dose-dependent manner by SBL in the cultures. In cultured PC12 cells, the expression of neurofilament, a protein marker for neuronal differentiation, was markedly induced by applied herbal extract. Moreover, the nerve growth factor- (NGF-) induced neurite outgrowth in cultured PC12 cells was significantly potentiated by the cotreatment of SBL. In accord, the expression of neurofilament was increased in the treatment of SBL. These results therefore suggested a possible role of SBL by its effect on neuron differentiation and protection against oxidative stress.

Neuroprotective Activity of Thioctic Acid in Central Nervous System Lesions Consequent to Peripheral Nerve Injury

Peripheral neuropathies are heterogeneous disorders presenting often with hyperalgesia and allodynia. This study has assessed if chronic constriction injury (CCI) of sciatic nerve is accompanied by increased oxidative stress and central nervous system (CNS) changes and if these changes are sensitive to treatment with thioctic acid. Thioctic acid is a naturally occurring antioxidant existing in two optical isomers (+)- and (−)-thioctic acid and in the racemic form. It has been proposed for treating disorders associated with increased oxidative stress. Sciatic nerve CCI was made in spontaneously hypertensive rats (SHRs) and in normotensive reference cohorts. Rats were untreated or treated intraperitoneally for 14 days with (+/−)-, (+)-, or (−)-thioctic acid. Oxidative stress, astrogliosis, myelin sheets status, and neuronal injury in motor and sensory cerebrocortical areas were assessed. Increase of oxidative stress markers, astrogliosis, and neuronal damage accompanied by a decreased expression of neurofilament were observed in SHR. This phenomenon was more pronounced after CCI. Thioctic acid countered astrogliosis and neuronal damage, (+)-thioctic acid being more active than (+/−)- or (−)-enantiomers. These findings suggest a neuroprotective activity of thioctic acid on CNS lesions consequent to CCI and that the compound may represent a therapeutic option for entrapment neuropathies.

Recurrent Laryngeal Nerve Regeneration through a Silicone Tube Produces Reinnervation without Vocal Fold Mobility in Rats

We established an animal model of recurrent laryngeal nerve reinnervation with persistent vocal fold immobility following recurrent laryngeal nerve injury. In 36 rats, the left recurrent laryngeal nerve was transected and the stumps were abutted in a silicone tube with a 1-mm interspace, facilitating regeneration. The mobility of the vocal folds was examined endoscopically 5, 10, and 15 weeks later. Electromyography of the thyroarytenoid muscle was performed. Reinnervation was assessed by means of a quantitative immunohistologic evaluation with anti-neurofilament antibody in the nerve both proximal and distal to the silicone tube. The atrophy of the thyroarytenoid muscle was assessed histologically. We observed that all animals had a fixed left vocal fold throughout the study. The average neurofilament expression in the nerve both distal and proximal to the silicone tube, the muscle area, and the amplitude of the compound muscle action potential recorded from the thyroarytenoid muscle on the treated side increased significantly (p < 0.05) over time, demonstrating regeneration through the silicone tube. Recurrent laryngeal nerve regeneration through a silicone tube produced reinnervation without vocal fold mobility in rats. The efficacy of new laryngeal reinnervation treatments can be assessed with this model.

Electrophysiological white matter dysfunction and association with neurobehavioral deficits following low-level primary blast trauma

There is strong evidence that primary blast injuries can cause neuropathological alterations in the brain. Clinical findings from war veterans indicating evidence of diffuse axonal injury have been corroborated by numerous primary blast models in animals. However, the effect of a subclinical blast (blast with no obvious sign of external trauma or lung injury) as a contributing factor to the neurological symptoms and neuropathology is less clear. Our group recently developed a model of low-level primary blast and characterized aberrant expression of white matter cytoskeletal proteins in the cortex and hippocampus following a subclinical wave shock exposure. Here we examined the susceptibility of the corpus callosum following subclinical blast. We also demonstrate that white matter dysfunction is associated with neurobehavioral deficits associated with anxiety and stress in rats. Anesthetized male Sprague–Dawley rats (~300g) were exposed to a primary blast (approx. 28kPa), below the threshold required to induce pulmonary trauma. Rats were evaluated on three behavioral outcome measures; the rotarod, the light/dark box and open field anxiety test. We used Western blotting to examine expression and degradation of axonally expressed αII-spectrin, NF200 and voltage-gated sodium channels (VGSC) in the corpus callosum. Acute slice preparations were used for electrophysiological analysis of evoked compound action potentials (CAPs) in the corpus callosum. There was evidence of αII-spectrin degradation in the corpus callosum at 48h post-injury detectable up to 14days post-injury, as well as increased heavy neurofilament expression. A reduction in VGSC expression was observed at 48h post-blast as well as a reduction in the interaction between ankyrin G and intact αII-spectrin. Blast exposed rats had significantly lower rotarod latency times relative to sham rats (p=0.002). Increased anxiety-related and stress-related behavior were observed in blast rats relative to sham animals as indicated by the increased frequency of fecal droppings (p=0.029) and reduced exploratory activity (p=0.036) in the open-field test. Blast rats had fewer transitions and time spent in lit sections of the light/dark box. Electrophysiological recordings from the corpus callosum indicated greater deficits in unmyelinated fibers of the corpus callosum relative to myelinated fibers characterized by reduced CAP amplitude response at 14days post-injury. Analysis of the relationship between stimulation distance to evoked response indicated an underlying abnormality in N1 myelinated fibers at close stimulation distances. Collectively, our results indicate that subclinical blast exposure can result in persistent neurological changes in cerebral white matter occurring in parallel with detectable neurobehavioral deficits.

Pulsed Radiofrequency Effects on the Lumbar Dorsal Root Ganglion of the Domestic Porcine: Pilot Study

SummaryIntroduction.Pulsed radiofrequency (PRF) is a percutaneous minimal invasive procedure for chronic pain management that can be used when conservative treatment methods have been ineffective. The effectiveness of PRF was demonstrated in various good quality randomized control studies, but mechanisms of action are still unclear.Aim of the Study.The aim of our study is to analyse the histological effects of PRF on the domestic porcine dorsal root ganglion (DRG), and evaluate the expression of biomarkers in gangliocytes of the subject(s).Materials and Methods.A total 3 domestic porcines were investigated. Under general anaesthesia and X-ray control, DRG PRF was performed. Four lumbar DRGs (L1, L2, L3, L4) were randomly treated. The opposite side DRGs was used as control. One month after the procedure the animal was euthanized. The lumbar region of the spine was placed in 10% formaldehyde for a month. After this fixation DRG samples were prepared for slide analysis. They were embedded in paraffin in order to obtain 3mm thick sections, which were then cut by microtome and collected on slide glasses. Using standard immunohistochemical reactions, the materials were tinted to define biomarkers neurofilaments (NF), glial fibrillary acidic protein (GFAP), heat shock protein - 70 (Hsp-70) expression and apoptosis by transferase-mediated dUTP nick-end labeling (TUNEL) analysis.Results.The number of cells with NF (26,0 ± 3,0 vs 16,1 ± 3,3; p&lt;0,05), GFAP (12,0 ± 1,3 vs 3,2 ± 0,9; p&lt;0,05) and Hsp-70 (10,0 ± 1,6 vs 4,2 ± 1,0; p&lt;0,05) expression, were larger in the PRF side comparing with the control side. Additionally, glial cells in spinal ganglia of both sides demonstrated immunoreactivity. The instances of apoptosis were not significantly different, in statistical terms, between the control and experimental sides (18,0 ± 4,0 vs 20,0 ± 4,0; p=0,35).Conclusions.PRF in spinal gangliocytes of lumbar region increases neural tissue cytoskeleton factors like NF and GFAP suggesting about active regeneration processes into the cells 1 month after the procedure. Spinal gangliocytes one month after PRF treatment notably increases Hsp-70 expression suggesting about activation of cellular activity and inhibitory role reducing of oxidative stress. Similar number of apoptotic cells in spinal ganglia of lumbar region after PRF and control side suggests about inhibitory role of PRF on programmed cell death and stimulation of cell survival.

Long-acting nanoformulated antiretroviral therapy elicits potent antiretroviral and neuroprotective responses in HIV-1-infected humanized mice

Long-acting nanoformulated antiretroviral therapy (nanoART) with improved pharmacokinetics, biodistribution and limited systemic toxicities will likely improve drug adherence and access to viral reservoirs. Atazanavir and ritonavir crystalline nanoART were formulated in a poloxamer-188 excipient by high-pressure homogenization. These formulations were evaluated for antiretroviral and neuroprotective activities in humanized NOD/scid-IL-2Rgc (NSG) mice. NanoART-treated NSG mice were evaluated for drug biodistribution, pharmacodynamics and toxicity. CD34 human hematopoietic stem cells were transplanted at birth in replicate NSG mice. The mice were infected with HIV-1ADA at 5 months of age. Eight weeks later, the infected animals were treated with weekly subcutaneous injections of nanoformulated ATV and RTV. Peripheral viral load, CD4 T-cell counts and lymphoid and brain histopathology and immunohistochemistry tests were performed. NanoART treatments by once-a-week injections reduced viral loads more than 1000-fold and protected CD4 T-cell populations. This paralleled high ART levels in liver, spleen and blood that were in or around the human minimal effective dose concentration without notable toxicities. Importantly, examination of infected brain subregions showed that nanoART elicited neuroprotective responses with detectable increases in microtubule-associated protein-2, synaptophysin and neurofilament expression when compared to untreated virus-infected animals. Therapeutic interruptions produced profound viral rebounds. Long-acting nanoART has translational potential with sustained and targeted efficacy and with limited systemic toxicities. Such success in drug delivery and distribution could improve drug adherence and reduce viral resistance in infected people.

Ginsenoside Rg1 facilitates neural differentiation of mouse embryonic stem cells via GR-dependent signaling pathway

Ginsenoside Rg1, a steroidal saponin of high abundance in ginseng, possesses the neuroprotective effects. In this study, we tried to explore the effect of Rg1 on promoting differentiation of mouse embryonic stem (ES) cells towards the neuronal lineage and its potential role involved in glucocorticoid receptor (GR) activation. Rg1 treatment induced a remarkable increase in the population of neuron-like cells in a time-dependent manner. More than 80% of Rg1-treated embryoid bodies (EBs) differentiated into neuron-like cells on d 8+10. Furthermore, the gradually increased protein expression of neurofilament (NEFM) and β-tubulin III (a neuronal specific protein) was determined. GR expression gradually increased during the differentiation course. RU486, an antagonist of GR, could efficiently block the neurogenesis-promoting activity of Rg1. On the other side, Rg1 stimulated the phosphorylation of ERK1/2 and Akt at different time points through GR activation-dependent mechanisms. Treatment of both U0126 (an inhibitor of MEK) and LY294002 (an inhibitor of PI3 K), hampered the neuronal differentiation induced by Rg1. Meantime, U0126 further decreased Rg1-induced p-Akt expression. In conclusion, Rg1 possesses the effects on inducing differentiation of mouse ES cells into neurons in vitro via the GR-MEK-ERK1/2-PI3 K-Akt signaling pathway.

Expression and significance of phosphorylated neurofilaments in hippocampus after caudoputamen hemorrhage in rat model

Objective To investigate the expression and significance of phosphorylated neurofilaments in the hippocampus after caudoputamen hemorrhage in a rat model.Methods Intracerebral hemorrhage model was established by injection of collagenase type Ⅶ into caudoputamen of rats.The neurological behavior scores of rats were assessed at different periods after the injection.The expression of phosphorylated neurofilaments in the hippocampus was detected by using Western blotting and immunohistochemistry.Results Rats showed neurological behavior deficit to varying degrees at different periods after caudoputamen hemorrhage (the averaged bederson score in 1-day operation group,3-day group and 5-day group was 0.9,1.5 and 2.0 respectively).The neurofilament phosphorylation in the hippocampus began to appear at 1st day after the injection and increased most obviously at 5th day.Conclusion The phosphorylated neurofilaments in the hippocampus after caudoputamen hemorrhage in the rat model may reflect the degree of neurological behavior deficit. Key words: Intracerebral hemorrhage; Caudoputamen; Hippocampal; Neurofilament phosphorylation

Spatio‐Temporal Development of Axonopathy in Canine Intervertebral Disc Disease as a Translational Large Animal Model for Nonexperimental Spinal Cord Injury

Spinal cord injury (SCI) represents a devastating central nervous system disease that still lacks sufficient therapies. Here, dogs are increasingly recognized as a preclinical animal model for the development of future therapies. The aim of this study was a detailed characterization of axonopathy in canine intervertebral disc disease, which produces a mixed contusive and compressive injury and functions as a spontaneous translational animal model for human SCI. The results revealed an early occurrence of ultrastructurally distinct axonal swelling. Immunohistochemically, enhanced axonal expression of β-amyloid precursor protein, non-phosphorylated neurofilament (n-NF) and growth-associated protein-43 was detected in the epicenter during acute canine SCI. Indicative of a progressive axonopathy, these changes showed a cranial and caudally accentuated spatial progression in the subacute disease phase. In canine spinal cord slice cultures, immunoreactivity of axons was confined to n-NF. Real-time quantitative polymerase chain reaction of naturally traumatized tissue and slice cultures revealed a temporally distinct dysregulation of the matrix metalloproteinases (MMP)-2 and MMP-9 with a dominating expression of the latter. Contrasting to early axonopathy, diminished myelin basic protein immunoreactivity and phagocytosis were delayed. The results present a basis for assessing new therapies in the canine animal model for translational research that might allow partial extrapolation to human SCI.

Leuprolide Acetate, a GnRH Agonist, Improves Experimental Autoimmune Encephalomyelitis: A Possible Therapy for Multiple Sclerosis

Gonadotrophin-releasing hormone (GnRH), a well known hypothalamic neuropeptide, has been reported to possess neurotrophic properties. Leuprolide acetate, a synthetic analogue of GnRH is considered to be a very safe and tolerable drug and it has been used for diverse clinical applications, including the treatment of prostate cancer, endometriosis, uterine fibroids, central precocious puberty and in vitro fertilization techniques. The present study was designed to determine whether Leuprolide acetate administration, exerts neurotrophic effects on clinical signs, body weight gain, neurofilaments (NFs) and myelin basic protein (MBP) expression, axonal morphometry and cell infiltration in spinal cord of experimental autoimmune encephalomyelitis (EAE) rats. In this work, we have found that Leuprolide acetate treatment decreases the severity of clinical signs of locomotion, induces a significantly greater body weight gain, increases the MBP and NFs expression, axonal area and cell infiltration in EAE animals. These results suggest the use of this agonist as a potential therapeutic approach for multiple sclerosis.

Cell specific ultrasound effects are dose and frequency dependent

Ultrasound is widely used in clinical practice, mostly in diagnostic studies, but increasingly in therapeutic applications as well. This may be the case in acceleration of wound healing or treatment of cancer. Still, little is known about the direct effect of frequency or energy density of the ultrasound upon the cells themselves. We therefore investigated the impact of three different protocols using high, medium and low energy densities at three different frequencies on normal endothelial and epithelial as well as carcinoma cell lines (neuroblastoma and adenocarcinoma cell lines).Proliferation of endothelial and epithelial cell lines was significantly increased depending on the frequency and energy density applied. No influence on actin cytoskeleton formation was seen in these cells after treatment, while a significant decrease in the density of microvilli and the length of filopodia in the epithelial cell line could be noted. The proliferation rate of the carcinoma cell lines was reduced and cells destroyed. Apoptosis was induced in the adenocarcinoma cells after ultrasound exposure. Additionally, the expression of neurofilament was increased in neuroblastoma cells as evidence of beginning differentiation.So, different settings of frequency and energy density in an ultrasonic treatment protocol lead to different impacts on proliferation, morphology and differentiation and might be used to stimulate or inhibit the growth of individual cell types.

Gonadotropin-releasing hormone treatment improves locomotor activity, urinary function and neurofilament protein expression after spinal cord injury in ovariectomized rats

It was reported that the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH) produces neurotrophic effects and that the spinal cord possesses GnRH receptors. The aim of the present study was to determine whether administration of GnRH improves locomotor activity, urinary function and neurofilament (NFs) protein expression after spinal cord injury (SCI) in ovariectomized rats. SCI was induced by balloon inflation model resulting in paraplegia. Locomotion was evaluated according to the Basso, Beattie, and Bresnahan Scale. Rats were subjected to bladder compression, twice daily until bladder reflex was established. NFs of 68, 160 and 200kDa from spinal cords were analyzed by electrophoresis. GnRH (60μg/kg) or physiologic NaCl solution was administered at 1 day after SCI and then daily for 15 days and the functional evaluation was realized for 5 weeks. Our results indicate that locomotor activity, restoration of urinary dysfunction and NFs expression of 160 and 200kDa were improved in SCI animals given GnRH compared to those without treatment. These findings suggest that GnRH acts as a neurotrophic factor and may be used as a potential therapeutic agent for treatment of SCI.

Inhibitory Activity of Bevacizumab to Differentiation of Retinoblastoma Cells

Vascular endothelial growth factor (VEGF) is a major regulator in retinal and choroidal angiogenesis, which are common causes of blindness in all age groups. Recently anti-VEGF treatment using anti-VEGF antibody has revolutionarily improved the visual outcome in patients with vaso-proliferative retinopathies. Herein, we demonstrated that bevacizumab as an anti-VEGF antibody could inhibit differentiation of retinoblastoma cells without affection to cellular viability, which would be mediated via blockade of extracellular signal-regulated kinase (ERK) 1/2 activation. The retinoblastoma cells expressed VEGFR-2 as well as TrkA which is a neurotrophin receptor associated with differentiation of retinoblastoma cells. TrkA in retinoblastoma cells was activated with VEGF treatment. Interestingly even in the concentration of no cellular death, bevascizumab significantly attenuated the neurite formation of differentiated retinoblastoma cells, which was accompanied by inhibition of neurofilament and shank2 expression. Furthermore, bevacizumab inhibited differentiation of retinoblastoma cells by blockade of ERK 1/2 activation. Therefore, based on that the differentiated retinoblastoma cells are mostly photoreceptors, our results suggest that anti-VEGF therapies would affect to the maintenance or function of photoreceptors in mature retina.