Hamilton Syringe Research Articles

RP-HPLC AND HPTLC METHODS FOR THE ESTIMATION OF RILUZOLE IN TABLET DOSAGE FORM

Two simple, specific, accurate and precise methods, namely, Reverse Phase High-Performance Liquid Chromatography and high-performance thin layer chromatography were developed for estimation of Riluzole in tablet dosage form. For the HPLC method, Promosil C-18, 5 μm column consisting of 200 × 4.6 mm i.d in isocratic mode, with mobile phase containing 25 mM KH2PO4 buffer (pH 2.8) : acetonitrile: (40:60 v/v) was used. The flow rate was 1 ml/min and effluent was monitored at 265 nm. The retention time was found to be 6.7 min. For the high-performance thin layer chromatographic method a Camag system comprising of Linnomat V automatic sample applicator, Hamilton syringe, Camag TLC Scanner-3, Camag WinCATs software with stationary phase precoated silica gel 60F254 and mobile phase consisting of chloroform: ethyl acetate: methanol (4:5.9:0.1 v/v/v) were used. The detection of spot was carried out at 265 nm. The Rf value was found to be 0.75. The methods were validated in terms of linearity, accuracy and precision. The linearity curves were found to be linear over 35-115 μg/ml for high-performance thin layer chromatography and 100-900 ng/spot for high-performance thin layer chromatography. The proposed methods were successfully used for estimation of Riluzole in tablet dosage form.

Rapid Plasticity of Visual Responses in the Adult Lateral Geniculate Nucleus

Compared to the developing visual system, where neuronal plasticity has been well characterized at multiple levels, little is known about plasticity in the adult, particularly within subcortical structures. We made intraocular injections of 2-amino-4-phosphonobutyric acid (APB) in adult cats to block visual responses in On-center retinal ganglion cells and examined the consequences on visual responses in the lateral geniculate nucleus (LGN) of the thalamus. In contrast to current views of retinogeniculate organization, which hold that On-center LGN neurons should become silent with APB, we find that ∼50% of On-center neurons rapidly develop Off-center responses. The time course of these emergent responses and the actions of APB in the retina indicate the plasticity occurs within the LGN. These results suggest there is greater divergence of retinogeniculate connections than previously recognized and that functionally silent, nonspecific retinal inputs can serve as a substrate for rapid plasticity in the adult.

Development of new models of orthotopic primary human retinoblastoma xenografts

AbstractPurpose To develop preclinical orthotopic models of primary human retinoblastomaMethods Orthotopic models of human retinoblastoma have been developed from three subcutaneous xenografted models that have previously been established and characterized in our laboratory, i.e. RB102‐FER, RB111‐MIL, and RB200‐GS that have been maintained in nude mice. Mice bearing xenografts were sacrificed and tumors were dissected to obtain a suspension of fresh tumor cells at a concentration of 8000 cells/l in DMEM serum‐free medium. Under intraperitoneal anesthesia, 2l of cell suspension was injected into the subretinal space of the right eye for 3 groups of mice using a 32G needle via a Hamilton syringe. Each group was constituted by 2 female SQUID mice and 3 male nude mice. After subretinal injection, ophthalmic examination of the mice was done every 15 days with binocular microscope. When tumor cells invaded vitreal cavity and anterior chamber, the mice were sacrificed for ophthalomological pathological analyses.Results Tumor cells developed in all injected eyes, whatever the mouse categories used, 4 weeks after orthotopic transplantation for RB102‐FER and 6 weeks after for RB200‐GS. In contrast, no tumor growth was observed in injected eyes of the RB111‐MIL model. In the 2 first xenografts, the proliferation started with a white hemorrhagic retinal mass; three weeks later, the vitreous was invaded by tumor cells that extended to the anterior chamber. Pathological examination of the injected eyes confirmed the presence of a massive infiltration of the retina, vitreous and anterior chamber by retinoblastoma cells.Conclusion We have then developed two new models of orthotopic primary human retinoblastoma that well reproduce the patient’s tumors.

Switching control of sympathetic activity from forebrain to hindbrain in chronic dehydration

We investigated the mechanisms responsible for increased blood pressure and sympathetic nerve activity (SNA) caused by 2-3 days dehydration (DH) both in vivo and in situ preparations. In euhydrated (EH) rats, systemic application of the AT(1) receptor antagonist Losartan and subsequent pre-collicular transection (to remove the hypothalamus) significantly reduced thoracic (t)SNA. In contrast, in DH rats, Losartan, followed by pre-collicular and pontine transections, failed to reduce tSNA, whereas transection at the medulla-spinal cord junction massively reduced tSNA. In DH but not EH rats, selective inhibition of the commissural nucleus tractus solitarii (cNTS) significantly reduced tSNA. Comparable data were obtained in both in situ and in vivo (anaesthetized/conscious) rats and suggest that following chronic dehydration, the control of tSNA transfers from supra-brainstem structures (e.g. hypothalamus) to the medulla oblongata, particularly the cNTS. As microarray analysis revealed up-regulation of AP1 transcription factor JunD in the dehydrated cNTS, we tested the hypothesis that AP1 transcription factor activity is responsible for dehydration-induced functional plasticity. When AP1 activity was blocked in the cNTS using a viral vector expressing a dominant negative FosB, cNTS inactivation was ineffective. However, tSNA was decreased after pre-collicular transection, a response similar to that seen in EH rats. Thus, the dehydration-induced switch in control of tSNA from hypothalamus to cNTS seems to be mediated via activation of AP1 transcription factors in the cNTS. If AP1 activity is blocked in the cNTS during dehydration, sympathetic activity control reverts back to forebrain regions. This unique reciprocating neural structure-switching plasticity between brain centres emphasizes the multiple mechanisms available for the adaptive response to dehydration.

A micro-advancer device for vitreal injection and retinal recording and stimulation

A micro-advancer device that positions a narrow-gauge needle within the vitreous humor of the rat eye is described. The device is compact, simple and inexpensive to manufacture. It consists of an outer guard needle and an inner injection needle that is advanced through the guard needle. With the rat held in a stereotaxic holder and the globe fixed to a stabilizing ring, the outer 25-gauge guard needle is advanced through the sclera using a standard micromanipulator. The inner 31-gauge injection needle is then advanced through the guard needle with a manually controlled leadscrew and carriage mechanism. The inner injection needle is attached to a Hamilton syringe and can be positioned to within microns of the retinal surface under visual observation through a microscope. The injection needle is fixed to the device by a quick-release clamp on the carriage and can be rapidly exchanged while the guard needle remains in place in the vitreous. This permits different solutions to be injected sequentially into the vitreous humor. Recording electrodes, stimulating electrodes, and optical fibers can also be advanced through the guard needle and positioned accurately near the retinal surface or within the retina.

Isolation of brain-infiltrating leukocytes.

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 μm, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1(+), CD11b(+) and F4/80(+)cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity

Excitotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-D-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca(2+)-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca(2+)](i)) increase. However, the basal level of [Ca(2+)](i) is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca(2+)](i) with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron.

Naturally Secreted Amyloid-β Increases Mammalian Target of Rapamycin (mTOR) Activity via a PRAS40-mediated Mechanism

Reducing the mammalian target of rapamycin (mTOR) activity increases lifespan and health span in a variety of organisms. Alterations in protein homeostasis and mTOR activity and signaling have been reported in several neurodegenerative disorders, including Alzheimer disease (AD); however, the causes of such deregulations remain elusive. Here, we show that mTOR activity and signaling are increased in cell lines stably transfected with mutant amyloid precursor protein (APP) and in brains of 3xTg-AD mice, an animal model of AD. In addition, we show that in the 3xTg-AD mice, mTOR activity can be reduced to wild type levels by genetically preventing Aβ accumulation. Similarly, intrahippocampal injections of an anti-Aβ antibody reduced Aβ levels and normalized mTOR activity, indicating that high Aβ levels are necessary for mTOR hyperactivity in 3xTg-AD mice. We also show that the intrahippocampal injection of naturally secreted Aβ is sufficient to increase mTOR signaling in the brains of wild type mice. The mechanism behind the Aβ-induced mTOR hyperactivity is mediated by the proline-rich Akt substrate 40 (PRAS40) as we show that the activation of PRAS40 plays a key role in the Aβ-induced mTOR hyperactivity. Taken together, our data show that Aβ accumulation, which has been suggested to be the culprit of AD pathogenesis, causes mTOR hyperactivity by regulating PRAS40 phosphorylation. These data further indicate that the mTOR pathway is one of the pathways by which Aβ exerts its toxicity and further support the idea that reducing mTOR signaling in AD may be a valid therapeutic approach.

Ameliorative effect of NAP on laser-induced retinal damage

NAP is the smallest active element of activity-dependent neuroprotective protein (ADNP) in the non-myelinated neural tissue. This study evaluated the neuroprotective effect of NAP in reducing the spread of laser-induced retinal damage in rat. Laser lesions were created in 72 DA pigmented rats. Two groups were treated by one intravenous or intravitreal injection of NAP immediately after exposure to laser. Two control groups were similarly administered saline injection. Histological and morphometrical evaluations of the lesions were preformed 3, 20 and 60 days after photocoagulation. After intravitreal treatment with NAP, a significant reduction in the diameter of the laser-induced lesions was found 3 days after photocoagulation (p < 0.001) but not after 20 and 60 days while the systemic treatment significantly reduced lesion diameter 20 and 60 days after photocoagulation (p = 0.001). Significant difference in photoreceptor cell loss was found in eyes treated intravitreally only 3 days after photocoagulation (p = 0.002). In the systemically treated animals such effect was found only after 20 and 60 days (p < 0.001). Treatment with NAP ameliorates laser-induced retinal lesions. Intravitreal treatment had an early short-term effect while the effect of systemic administration was delayed and prolonged. This treatment may be of clinical significance in reducing laser-induced retinal injuries in humans.

Effects of Selective iNOS Inhibitor on Spatial Memory in Recovered and Non-Recovered Ketamine Induced Anesthesia.

Nitric oxide (NO) is thought to be involved in spatial learning and memory in several brain areas such as hippocampus. This study examined the effects of post-training intrahippocampal microinjections of 1400W as a selective iNOS inhibitor on spatial memory, in anesthetized and non-anesthetized situations in rats. In the present work, 4-day training trials of animals were conducted. Spatial memory was tested 48 hours after the drug infusions. For microinjection of 1400W into CA1 region of the hippocampus in conscious animals, guide cannula was implanted into the CA1 area and 1400W was infused after recovery from surgical anesthesia. In anesthetized animals, 1400W was microinjected directly into CA1 region by Hamilton syringe during anesthesia. After completion of training, 1400W (10, 50 and 100 μM/side) were microinjected bilaterally (1 μL/side) and testing trials were performed 48 h after drug infusions in both groups of cannulated and non-cannulated rats. Significant reduction was observed in escape latency and traveled distance in animals that received 1400W (100 μM/side, *p < 0.05) via cannula after recovery in comparison with control group. Also, microinjection of 1400W (100 μM/side) in post recovery phase caused a significant (***p < 0.001) reduction in time and distance of finding the hidden platform in comparison with anesthetized situation. These findings suggest that 1400W has a significant improvement on spatial memory and memory enhancement induced by iNOS inhibitor can be affected by anesthesia in a period of time.

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2β (VIA)-deficient mice

Ca(2+)-independent phospholipase A(2)β (iPLA(2)β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA(2)β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M(1,3,5) receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA(2)β(-/-), iPLA(2)β(+/-), and iPLA(2)β(+/+) mice, and [1-(14)C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J(in), representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA(2)β(-/-) or iPLA(2)β(+/-) compared with iPLA(2)β(+/+) mice showed widespread and significant baseline reductions in k* and J(in) for DHA. Arecoline increased both parameters in brain regions of iPLA(2)β(+/+) mice but quantitatively less so in iPLA(2)β(-/-) and iPLA(2)β(+/-) mice. Consistent with iPLA(2)β's reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA(2)β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M(1,3,5) receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.

Reduction of Brain β-Amyloid (Aβ) by Fluvastatin, a Hydroxymethylglutaryl-CoA Reductase Inhibitor, through Increase in Degradation of Amyloid Precursor Protein C-terminal Fragments (APP-CTFs) and Aβ Clearance

Epidemiological studies suggest that statins (hydroxymethylglutaryl-CoA reductase inhibitors) could reduce the risk of Alzheimer disease. Although one possible explanation is through an effect on beta-amyloid (Abeta) metabolism, its effect remains to be elucidated. Here, we explored the molecular mechanisms of how statins influence Abeta metabolism. Fluvastatin at clinical doses significantly reduced Abeta and amyloid precursor protein C-terminal fragment (APP-CTF) levels among APP metabolites in the brain of C57BL/6 mice. Chronic intracerebroventricular infusion of lysosomal inhibitors blocked these effects, indicating that up-regulation of the lysosomal degradation of endogenous APP-CTFs is involved in reduced Abeta production. Biochemical analysis suggested that this was mediated by enhanced trafficking of APP-CTFs from endosomes to lysosomes, associated with marked changes of Rab proteins, which regulate endosomal function. In primary neurons, fluvastatin enhanced the degradation of APP-CTFs through an isoprenoid-dependent mechanism. Because our previous study suggests additive effects of fluvastatin on Abeta metabolism, we examined Abeta clearance rates by using the brain efflux index method and found its increased rates at high Abeta levels from brain. As LRP1 in brain microvessels was increased, up-regulation of LRP1-mediated Abeta clearance at the blood-brain barrier might be involved. In cultured brain microvessel endothelial cells, fluvastatin increased LRP1 and the uptake of Abeta, which was blocked by LRP1 antagonists, through an isoprenoid-dependent mechanism. Overall, the present study demonstrated that fluvastatin reduced Abeta level by an isoprenoid-dependent mechanism. These results have important implications for the development of disease-modifying therapy for Alzheimer disease as well as understanding of Abeta metabolism.

Effects of Selective iNOS Inhibitor on Spatial Memory in Recovered and Non-recovered Ketamine Induced-anesthesia in Wistar Rats.

Nitric oxide (NO) is thought to be involved in spatial learning and memory in several brain areas such as hippocampus. This study examined the effects of post-training intrahippocampal microinjections of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor on spatial memory, in both anesthetized and non-anesthetized situations in rats. In the present work, 4-day training trials of animals were conducted. Spatial memory was tested 48 h after the drug infusions. For microinjection of 1400W into CA1 region of the hippocampus in conscious animals, guide cannula was implanted into the CA1 area and 1400W was infused after recovery from surgical anesthesia. In anesthetized animals, 1400W was microinjected directly into CA1 region by Hamilton syringe during anesthesia. After completion of training, 1400W (10, 50 and 100 μM/side) were microinjected bilaterally (1 μL/side) and testing trials were performed 48 h after drug infusions in both groups of cannulated and non-cannulated rats. Significant reduction was observed in escape latency and traveled distance in animals that received 1400W (100 μM/side, * P < 0.05) via cannula after recovery in comparison with control group. Moreover, microinjection of 1400W (100 μM/side) in post recovery phase also caused a significant (*** P < 0.001) reduction in time and distance of finding the hidden platform in comparison with anesthetized situation. These results suggest that 1400W has a significant improvement on spatial memory, and memory enhancement induced by iNOS inhibitor can be affected by anesthesia in a period of time.

Involvement of Extracellular Superoxide Dismutase in Regulating Brain Blood Flow

The physiological role of extracellular superoxide dismutase (SOD3) has received insufficient study. We investigated the hypothesis that SOD3, which neutralizes superoxide anions (O2(-)) in the intercellular space of the brain, prevents the inactivation of nitric oxide (NO) and is thus involved in regulating cerebral vascular tone. Local brain blood flow was measured in the striatum of anesthetized rats during administration of various combinations of a SOD mimetic, a SOD inhibitor, an NO donor, and an NOS inhibitor into the striatum using a Hamilton syringe. In normal conditions, SOD3 was found to minimize O2(-) levels, protecting endogenously produced NO at a sufficient level to maintain cerebral vascular tone and reactivity. SOD3 was found to increase the vasodilatory effect of endogenously produced NO in the brain. SOD3 was found to neutralize superoxide anions produced in the brain during respiration of 100% O2 and to maintain basal NO levels and its vasodilatory potential in normobaric hyperoxia.

Neuroprotective Effect of Oligodendrocyte Precursor Cell Transplantation in a Long-Term Model of Periventricular Leukomalacia

Perinatal white matter injury, or periventricular leukomalacia (PVL), is the most common cause of brain injury in premature infants and is the leading cause of cerebral palsy. Despite increasing numbers of surviving extreme premature infants and associated long-term neurological morbidity, our understanding and treatment of PVL remains incomplete. Inflammation- or ischemia/hypoxia-based rodent models, although immensely valuable, are largely restricted to reproducing short-term features of up to 3 weeks after injury. Given the long-term sequelae of PVL, there is a need for subchronic models that will enable testing of putative neuroprotective therapies. Here, we report long term characterization of a neonatal inflammation-induced rat model of PVL. We show bilateral ventriculomegaly, inflammation, reactive astrogliosis, injury to pre-oligodendrocytes, and neuronal loss 8 weeks after injury. We demonstrate neuroprotective effects of oligodendrocyte precursor cell transplantation. Our findings present a subchronic model of PVL and highlight the tissue protective effects of oligodendrocyte precursor cell transplants that demonstrate the potential of cell-based therapy for PVL.

SOCS3 Deletion Promotes Optic Nerve Regeneration In Vivo

Axon regeneration failure accounts for permanent functional deficits following CNS injury in adult mammals. However, the underlying mechanisms remain elusive. In analyzing axon regeneration in different mutant mouse lines, we discovered that deletion of suppressor of cytokine signaling 3 (SOCS3) in adult retinal ganglion cells (RGCs) promotes robust regeneration of injured optic nerve axons. This regeneration-promoting effect is efficiently blocked in SOCS3-gp130 double-knockout mice, suggesting that SOCS3 deletion promotes axon regeneration via a gp130-dependent pathway. Consistently, a transient upregulation of ciliary neurotrophic factor (CNTF) was observed within the retina following optic nerve injury. Intravitreal application of CNTF further enhances axon regeneration from SOCS3-deleted RGCs. Together, our results suggest that compromised responsiveness to injury-induced growth factors in mature neurons contributes significantly to regeneration failure. Thus, developing strategies to modulate negative signaling regulators may be an efficient strategy of promoting axon regeneration after CNS injury.

Hypercholesterolemic Mice Exhibit Lymphatic Vessel Dysfunction and Degeneration

Lymphatic vessels are essential for lipid absorption and transport. Despite increasing numbers of observations linking lymphatic vessels and lipids, little research has been devoted to address how dysregulation of lipid balance in the blood, ie, dyslipidemia, may affect the functional biology of lymphatic vessels. Here, we show that hypercholesterolemia occurring in apolipoprotein E-deficient (apoE(-/-)) mice is associated with tissue swelling, lymphatic leakiness, and decreased lymphatic transport of fluid and dendritic cells from tissue. Lymphatic dysfunction results in part from profound structural abnormalities in the lymphatic vasculature: namely, initial lymphatic vessels were greatly enlarged, and collecting vessels developed notably decreased smooth muscle cell coverage and changes in the distribution of lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1). Our results provide evidence that hypercholesterolemia in adult apoE(-/-) mice is associated with a degeneration of lymphatic vessels that leads to decreased lymphatic drainage and provides an explanation for why dendritic cell migration and, thus, immune priming, are compromised in hypercholesterolemic mice.

Interferon-γ Prevents Death of Bystander Neurons during CD8 T Cell Responses in the Brain

T cells restricted to neurotropic viruses are potentially harmful as their activity may result in the destruction of neurons. In the Borna disease virus (BDV) model, antiviral CD8 T cells entering the brain of infected mice cause neurological disease but no substantial loss of neurons unless the animals lack interferon-gamma (IFN-gamma). We show here that glutamate receptor antagonists failed to prevent BDV-induced neuronal loss in IFN-gamma-deficient mice, suggesting that excitotoxicity resulting from glutamate receptor overstimulation is an unlikely explanation for the neuronal damage. Experiments with IFN-gamma-deficient mice lacking eosinophils indicated that these cells, which specifically accumulate in the infected brains of IFN-gamma-deficient mice, are not responsible for CA1 neuronal death. Interestingly, BDV-induced damage of CA1 neurons was reduced significantly in IFN-gamma-deficient mice lacking perforin, suggesting a key role for CD8 T cells in this pathological process. Specific death of hippocampal CA1 neurons could be triggered by adoptive transfer of BDV-specific CD8 T cells from IFN-gamma-deficient mice into uninfected mice that express transgene-encoded BDV antigen at high level in astrocytes. These results indicate that attack by CD8 T cells that cause the death of CA1 neurons might be directed toward regional astrocytes and that IFN-gamma protects vulnerable CA1 neurons from collateral damage resulting from exposure to potentially toxic substances generated as a result of CD8 T cell-mediated impairment of astrocyte function.