In vivo visualisation of nanoparticle entry into central nervous system tissue.

Because the potential neurotoxicity of nanoparticles is a significant issue, characterisation of nanoparticle entry into the brain is essential. Here, we describe an in vivo confocal neuroimaging method (ICON) of visualising the entry of fluorescent particles into the parenchyma of the central nervous system (CNS) in live animals using the retina as a model. Rats received intravenous injections of fluorescence-labelled polybutyl cyanoacrylate nanoparticles that had been synthesised by a standard miniemulsion polymerisation process. We performed live recording with ICON from before and up to 9 days after particle injection and took photomicrographs of the retina. In addition, selective retrograde labelling of the retinal ganglion cells was achieved by stereotaxic injection of a fluorescent dye into the superior colliculus. Using ICON, we observed vascular kinetics of nanoparticles (wash-in within seconds), their passage to the retina parenchyma (within minutes) and their distribution (mainly cellular) under in vivo conditions. For the detection of cell loss--which is important for the evaluation of toxic effects--in another experiment, we semi-quantitatively analysed the selectively labelled retinal neurons. Our results suggest that the dye per se does not lead to neuronal death. With ICON, it is possible to study nanoparticle kinetics in the retina as a model of the blood-brain barrier. Imaging data can be acquired within seconds after the injection, and the long-term fate of cellular uptake can be followed for many days to study the cellular/extracellular distribution of the nanoparticles. ICON is thus an effective and meaningful tool to investigate nanoparticle/CNS interactions.

In vivo confocal neuroimaging (ICON): non-invasive, functional imaging of the mammalian CNS with cellular resolution.

With in vivo confocal neuroimaging (ICON), single retinal ganglion cells (RGCs) can be visualized non-invasively, repeatedly, in real-time and under natural conditions. Here we report the use of ICON to visualize dynamic changes in RGC morphology, connectivity and functional activation using calcium markers, and to visualize nanoparticle transport across the blood-retina barrier by fluorescent dyes. To document the versatility of ICON, we studied the cellular response to optic nerve injury, and found evidence of reversible soma swelling, recovery of retrograde axonal transport and a difference in calcium activation dynamics between surviving and dying RGCs. This establishes ICON as a unique tool for studying CNS physiology and pathophysiology in real-time on a cellular level. ICON has potential applications in different research fields, such as neuroprotection/regeneration, degeneration, pharmacology, toxicity and drug delivery.

Simultaneous Administration of Statins and Pioglitazone Limits Tumor Growth in a Rat Model of Malignant Glioma.

BACKGROUND/AIM: Statins are cholesterol reducers with considerable dose-dependent effect against glioma cells. The apoptotic effect could be increased by combining statins or by adding pioglitazone (PGZ). The last one is an anti-diabetic drug, an agonist of the peroxisome proliferator-activated receptor-gamma (PPARG). We used an animal model to test the effect of such combination in vivo and we investigated the changes on immunological processes. MATERIALS AND METHODS: Thirty-three rats (F344/DuCrl) were anesthetized and glioblastoma (F98) cells were implanted stereotactically. Animals were randomized into four groups: i) control (N=9); ii) intraperitoneal injection of PGZ 10 mg/kg/day (N=8); iii) oral administration of atorvastatin (ATVS) 40 mg/kg and lovastatin (LVS) 50 mg/kg (N=8); iv) oral administration of ATVS 40 mg/kg, LVS 50 mg/kg and PGZ 5 mg/kg (N=8). Treatment was started at 3rd postoperative day and continued for 14 days. The animals were followed-up for 30 days after start of therapy. Survival time, tumor volume, proliferation rate, counts of peripheral and tumor infiltrating leukocytes were compared. RESULTS: No difference of survival time or incidence of neurological deficits was observed. The combination of statins with PGZ led to a significant reduction in tumor volume by approximately 40% (p<0.05), statins combination was less effective and PGZ alone did not affect tumor volume. The groups treated with statins displayed significantly lower counts of peripheral CD3+, CD4+ and CD8+ T-cells and lower tumor associated CD68-positive cells (p<0.01, in respect to controls or PGZ alone). The proliferation rate was not statistically different. No relevant toxic effects were observed. DISCUSSION: Statins and PGZ are well-tolerated in rats and produced a significant tumor reduction, while an impact on neurological deficits or survival improvement could not be demonstrated. The reduction of infiltrating macrophages by using statins and PGZ should be further studied.

Brain restoration as an emerging field in neurology and neuroscience.

Restoration of brain function was long thought to be impossible. However, as the publications in the journal Restorative Neurology and Neuroscience (RNN) for more than 20 years attest, clinically useful improvement can be achieved after damage or diseases of the brain, the retina, and the peripheral nervous system. By reviewing both pre-clinical studies and clinical work, we explore what advancements can be made today and what to expect going forward. For example, in the last few years we have seen a clinical focus in the area of non-invasive brain stimulations and rehabilitation training trials. In basic animal research multi-modal approaches have been presented to restore brain function with a combination of different treatments. We think that this is an exciting time in the area of restoration of brain function with many new strategies aimed at helping recovering their impaired neurological functions.

Transcorneal electrical stimulation alters morphology and survival of retinal ganglion cells after optic nerve damage.

Traumatic optic nerve injury leads to retrograde death of retinal ganglion cells (RGCs), but transcorneal electrical stimulation (TES) can increase the cell survival rate. To understand the mechanisms and to further define the TES-induced effects we monitored in living animals RGC morphology and survival after optic nerve crush (ONC) in real time by using in vivo confocal neuroimaging (ICON) of the retina. ONC was performed in rats and ICON was performed before crush and on post-lesion days 3, 7 and 15 which allowed us to repeatedly record RGC number and size. TES or sham-stimulation were performed immediately after the crush and on post-injury day 11. Three days after ONC we detected a higher percentage of surviving RGCs in the TES group as compared to sham-treated controls. However, the difference was below significance level on day 7 and disappeared completely by day 15. The death rate was more variable amongst the TES-treated rats than in the control group. Morphological analysis revealed that average cell size changed significantly in the control group but not in stimulated animals and the morphological alterations of surviving neurons were smaller in TES-treated compared to control cells. In conclusion, TES delays post-traumatic cell death significantly. Moreover, we found "responder animals" which also benefited in the long-term from the treatment. Our in vivo cellular imaging results provide evidence that TES reduces ONC-associated neuronal swelling and shrinkage especially in RGCs which survived long-term. Further studies are now needed to determine the differences of responders vs. non-responders.

Transcorneal alternating current stimulation after severe axon damage in rats results in "long-term silent survivor" neurons.

Transcorneal alternating current stimulation (tACS) was proposed to decrease acute death of retinal ganglion cells after optic nerve transection in rats, but it is not known if cell survival is long-term and associated with functional restoration. We therefore evaluated the effects of tACS in a rat model of optic nerve crush using anatomical, electrophysiological and behavioural measures. Rats were trained in a brightness discrimination visual task and the retinal ganglion cell number was quantified with in vivo confocal neuroimaging. Thereafter, severe optic nerve crush or sham crush was performed and rats were treated under anaesthesia either with tACS or sham stimulation immediately after the lesion and on day 3, 7, 11, 15, 19 and 23. Brightness discrimination was evaluated for 6 weeks and retinal ganglion cells were counted in vivo on post-crush days 7 and 28. In additional rats we studied the influence of tACS on bioelectrical activity. On post-lesion day 28, the tACS-treated group showed a neuronal survival of 28.2% which was significantly greater than in sham operates (8.6%). All animals with optic nerve crush were significantly impaired in brightness discrimination and did not recover performance, irrespective to which group they belonged. In accordance with this, there was no significant influence of the stimulation on EEG power spectra. In conclusion, tACS induced long-term neuronal protection from delayed retrograde cell death, but in this case of severe axonal damage tACS did not influence functional restoration and EEG signals recorded over the visual cortex.

Recovery of axonal transport after partial optic nerve damage is associated with secondary retinal ganglion cell death in vivo.

PURPOSE: Traumatic injury of the optic nerve leads to retrograde cell death of retinal ganglion cells (RGCs) but usually a certain percentage of neurons survive. It has been suggested that recovery of axonal transport is beneficial for survival. The present study was therefore performed to provide a synopsis of the temporal pattern of axonal transport decline/recovery and the viability of RGCs after optic nerve crush (ONC). METHODS: Fluorescent dyes were injected into the superior colliculus to retrogradely label RGCs. Axonal transport kinetics into the RGCs was visualized with in vivo confocal neuroimaging (ICON) in uninjured rats and in rats which had mild or moderate ONC. Red fluorescent beads were injected on day 2 post-ONC and green beads on day 7. RESULTS: At 2 to 4 days post-ONC significant axonal transport was detected, but within 1 week the transport of the fluorescent beads was decreased. Interestingly, during post-ONC week 3 the axon transport slowly recovered. However, despite this recovery, retrograde cell death rate continued and was even increased in a "second wave" of cell death in those neurons that displayed axon transport recovery. CONCLUSIONS: After damage many surviving RGCs lose their axon transport, but after approximately 3 weeks, this transport recovers again, a sign of intrinsic axon repair. Contrary to the prediction, axon transport recovery is not associated with better cell survival but rather with a second wave of cell death. Thus, the accelerated cell death associated with recovery of axon transport suggests the existence of a late retrograde cell death signal.

Experience-dependent plasticity and vision restoration in rats after optic nerve crush.

Within the first weeks after brain damage the visual system can spontaneously recover, but it is not known if visual experience plays a role in this process. Therefore, we studied the role of visual experience during recovery by exposing rats to normal, enriched, and impoverished visual conditions. Adult rats, which had learned a six-choice brightness discrimination task, received bilateral partial optic nerve crush (ONC), and were then exposed daily for 34 days to either (1) total darkness, (2) a standard 12-h light:12-h dark cycle, or (3) a 2-h selective visual enrichment. The percentage of correct choices and the maximal performance levels reached were used as recovery end-points. Retinal ganglion cell (RGC) morphology was evaluated following retrograde transport of fluorescent tracer with in vivo confocal neuroimaging (ICON) before and after ONC. Whereas rats kept under normal daylight conditions after ONC recovered their visual functions rather well, rats housed in complete darkness did not recover at all. However, only 2 h of daily exposure to visual enrichment induced significantly enhanced recovery, which was even faster than that seen in rats housed under normal daylight conditions. RGC soma size changes were observed after ONC, but they did not correlate with any measures of behavioral recovery. We conclude that visual experience, even if provided for short daily periods, is a critical factor determining the dynamics of early phases of recovery.